Cephalexin 500 mg for clymidia

Elevate Pay

2024.01.23 12:33 steevo Elevate Pay

Unofficial Sub of elevatepay.co
[link]


2016.02.16 01:43 pookie_wocket Awesome sci-fi & fantasy book covers

The good twin of badscificovers. Celebrating the art of Sci-Fi paperbacks and pulp! Post your favorite Science-Fiction and Fantasy artists and covers here.
[link]


2018.04.18 04:19 DrMarioBrother ULDN

This subreddit is primarily focused on the microdosing of naltrexone, but naltrexone discussion of any kind is welcome. ULDN Ultra Low Dose Naltrexone Post about ULDN (typically ~0.5-200ug/day, <500ug for sure), LDN (Low Dose Naltrexone, often ~0.5-10mg/day), or even "normal" naltrexone doses (25-50mg/day).
[link]


2024.05.18 23:21 I_read_reddits_rules You're in a show like N and Afraid, but in this case, if you win, you get $100 000. You get to choose one of these options of things to help you. What do you choose? (click for details)

It's you and 8 others. The only things you are given as a group is a 2-gallon pot and a machete.
Each of you rolled dice and you won—you won the choice of any of these 6 options.
No one's allowed to take what you'd choose from you without your permission—you can use, lend, give away, and/or bargain with such as you wish. If anyone steals from you wt:thon would be kicked out by the show's producer(s).
The area is not quite a jungle, nor a desert, but somewhat in-between.
The game is for 3 months. If you make to then, or be the last standing if none of you do (minimally after a month), you get $100 000.

option 6:
10 liters of sunscreen, a 1 - 15 year-old bottle of aspirin (500 - 500 mg tablets), a 60 x 60 x 20 cm block of ice, and a charged electric drill (no bits).
View Poll
submitted by I_read_reddits_rules to polls [link] [comments]


2024.05.18 23:18 Ill-Switch74 Looking for my last partner

Looking for my last partner
3/4 are done and hoping to finish tonight please
submitted by Ill-Switch74 to monopolygo_fairtrade [link] [comments]


2024.05.18 22:39 JadedBarber5363 [WTS] [WTT] Slabs, 1/10 Gold Phil, Proof Sets, Ike’s, 10 oz Bars, 1 oz Bars, 1 oz Rounds/Coins, Kooks, Geigers, Fractional, Foreign, Goldbacks, Casino Tokens, Collectables, and more.

*Chat Preferred. Only accepting trades for items under “COLLECTABLES”. Items are verified using SIGMA Investor (thru and gravity), Pocket Pinger, Weight, and magnet.
[PROOF](https://imgur.com/a/qK4PKfz
GOLD
ROUNDS AND COINS:
2.5 Gram Silver Bar- $3
5x 1/10 Silver- $25
1/4 Oz Round- $10
1922 Peace Dollar- $26
1 oz APMEX Bar- $32
2000 The Dawn of A New Millennium 1oz Silver- $35
1 Gram Geiger In Assay- $10
40% lot ($5 Face)- $47
35% Lot (5 War Nickels)- $8.50
16x 1 oz Silver Towne Mighty Eagles- $512
4x 1 oz Buffalos- $128
7x 1 oz Misc Nicer Rounds- $230
4x Casino Tokens- $85 For All or $22 EA
5 oz Sealed Bar- $158
Cale Yarborough 1 oz Round- $45
1978 Cien Pesos- $20
4 Standing Liberty Quartahhhs- $25
1974 1 oz Liberty Medallion- $35
20x 1 oz Tokelau 2024 Proof Mustang- $36 EA for all for $34 EA
Numismatic 90% (1905 Barber Dime, 1915 Barber Quarter, 1901 O Barber Half, and 1937 Walker.)- $47
$9.50 Face 90% Lot ($6 JFK, $.50 Franklin, $1 Wash, $1.20 Rosevelt’s, $.80 Mercs all with dates and some are higher grades)- $208
2x 1962 Proof Sets (Some missing random pieces like cardboard or paper)- $26 EA
Blue Ike (1x 1971, 2x 1972, 3x 1974)- $13 Ea
10 oz Bar- $315
1 oz Peace Dollar Round- $40
5x 2023 Kookaburra (W/Capsule)- $35 EA
5x 2024 Kookaburra (W/Capsule)- $35 EA
1 oz Germania Mint Allegories (W/COA)- $45
1 oz 2024 BU T2 ASE- $35
500 Gram The Holy Mint Bar- $525
6x Vintage Bars (Engelhard, JM, and Silver Towne) Lot- $215
1964 Accented Hair Proof Set (Complete)- $70
24x 2024 1/10 Silver Brittanias- $10 EA or all for $9 EA
2001 S Proof JFK Half- $12
Foreign lot In Flips (31 Coins)- $25
Foreign lot Loose (21 Coins and most are Canadian)- $15
1 New Penny (26 coins dates all in the 1970s), 1/2 New Penny (16 coins dates all in the 1970s), and 2 New Pence Lot (26 coins dates all in the 1970s)- $40
1970 1 Piso Pope Paul VI (Non PM)- $5
1966 Jamaican Commonwealth Games Coin- $4
1983 Netherland Coin Set (Cracked Open Case)- $7
RANDOMS:
5x 1 Gram Argor Palladium- $65 EA
Silver 1975 Philippines Proof Set (complete)- $90
10 Oz “The Wedge” DHF Silver Bar- $380
SLABS: (Whale deal is buy all slabs get the 1900 Morgan for $60)
2022 1oz Serbia Nikola Tesla Natural Healing NGC MS70- $115
1950 S Booker T Washington PCGS MS65- $65
1944 Winged Liberty Dime PCGS MS66- $40
1964 Washington Quarter NGC PF67 (Crack on slab)- $15
1946 Walking Liberty Half PCI MS65- $95
2017 S ASE NGC PF70 Ultra Cameo San Fransico Slab- $120
1922 D Peace NGC MS63- $85
1900 Morgan PCGS MS63 (Toned)- $115
1883 Morgan NGC “Fattie” MS64 (Toned)- $125
2023 Morgan CAC First Delivery MS69 Slab- $100
Bradford Exchange 2023 T2 BU ASE- $40
1855 Germany 1 S Hamburg MS63 NGC (Toner)- $80
2007-S PCGS PR70DCAM John Adams- $11
COLLECTABLES: Will trade for low premium Silver and Gold. [WTT] [WTS]
2x 2021 Cameroon Crucifix Coin- $120 EA
6x Fiji 2022 Snowflake Coin W/Crystals (Sealed)- $85 EA
3x Fiji 2023 Wonders Of Nature Great Barrier Reef Coin- $145 EA
2021 Star Wars IG-11 NIUE Coin- $105
2021 Samoa Splash Of Colour (Color 🇺🇸) New York City Coin- $175
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Shipping/payment:
🚛*Shipping- is $5 for 1-8 oz (Ground Advantage), $6 for 9-12 oz (Ground Advantage) and $10 for over 12 oz (Priority Mail) both with tracking numbers. American shipping only 🇺🇸 (prices are for lower 48). I am shipping out of Oklahoma, if you are close I may be able to ship cheaper ground than priority on higher weights. Be sure to inquire if you expect that to be the case.
🧾*Payment- I accept Zelle (preferred), Venmo, cash app, and PayPal FF (least preferred and not available for low flair users). Please no notes/comments or I will refund (if you are forced to use one add an emoji or . ) Thanks!
💂 *Security- I will not give my password to anyone for any reason. I have enabled two factor authentication and have other steps to help protect myself and the buyers. I also use the same setup to take proof photos, so any deviation in that is a red flag 🚩.
submitted by JadedBarber5363 to CoinSales [link] [comments]


2024.05.18 22:04 Calm_Instruction1651 Rear LED Tail Light replacement

Is replacing the entire rear LED assembly the only option?
I'm finding REAL OEM a bit confusing. I think it's part #63216920699 for the drivers side rear.
Prices seem to range around $500 for OEM part. Any good DIY's?
Thank you
submitted by Calm_Instruction1651 to E46M3 [link] [comments]


2024.05.18 21:17 Wolfy0084 took Levofloxacin 500Mg 2 different times in 24 days and now feeling bad side effects.

So I am 28 (M) who took Levofloxacin for a UTI and felt fine for few days.
But then into the next few days I started to sleep fo about 2 or 3 hours a night. Then each day starts to fell hard to sleep at all. As days go on my vision becomes weird as hard to see far away and light start to become sensitive.
Few more days it seems I lost the stage of 3rd and 4th stages of sleep and with out maltoine and Hydroxyzine it's hard to sleep at all.
My new symptoms are now full blown body shakes and right leg starts small ting pain.
Also over the days am having memory problems
It caused my anxiety super high now to the point I feel like am dying or have a serious disease or something or feels like it cause me to think am starting to have nerve damage in my body..
Question also is this normal in a 24 day spand after taking (X2)500 MG of Levofloxacin and having the bad side effects???
submitted by Wolfy0084 to floxies [link] [comments]


2024.05.18 20:00 qckpckt 1989 Yamaha MG-III. A real sleeper guitar.

1989 Yamaha MG-III. A real sleeper guitar.
Some stats:
  • 26(!) frets
  • surprisingly excellent (noiseless!) pickups
  • push-push coil split on the bridge
  • unique body shape that (I think, anyway) that lends itself to a lot of genres.
  • made in Japan
  • relatively easy to find for ~$500 CAD
I’ve been on the hunt for an MG for a few years. Ideally I wanted an MG-K (white with 3 slanted single coils and a locking tremolo), but they’re a little harder to find. I actually quite like the scratch plate on this shape.
This is a solid guitar. It’s a two piece neck but the fretwork was great and the overall construction is great. High quality gotoh parts throughout.
This particular one had been set up with a blocked tremolo for probably most of the 35 years of its life, and this has compromised the knife edge of the tremolo fulcrum so it doesn’t have a single balance point.
By a huge stroke of luck, I had an identical NOS Gotoh tremolo unit in silver, so I have swapped that in until I can find the time to restore the original tremolo.
I had big plans for this guitar - refinishing it in a pastel or glow in the dark paint, creating a hew scratch plate, running it SSS… but frankly it’s in such fantastic shape that it would be a real shame to alter it.
These were only available in the Japanese domestic market, but they do show up on reverb every now and again with international shipping. There were later models that had only 22 frets. These for some reason seem to fetch higher prices.
Here’s a webpage with more details of the MG range.
submitted by qckpckt to guitars [link] [comments]


2024.05.18 19:44 Familiar_Egg_5362 Is hydrolyzed fish collagen peptides safe during pregnancy?

Is hydrolyzed fish collagen peptides safe during pregnancy?
I recently came across multi collagen peptides from micro ingredients on TikTok. I’m currently in my 1st trimester and would like to take this but I need to know if it safe to consume while pregnant? Help?
submitted by Familiar_Egg_5362 to pregnancy_care [link] [comments]


2024.05.18 19:03 sprout-andthebean Leukocytes (white blood cells) in urine / sterile pyuria

I had a routine blood and urine test and it came back slightly higher than normal for lymphocytes in my blood; leukocytes (white blood cells) and bacteria in my urine. My doctor made me retake the urine test and it came back with just leukocytes.
I don't feel like I have/had a UTI, though that would be great if it turns out to be that simple. I have no back pain, so my doctor doesn't think I have a kidney problem. I'm waiting to hear back from the urologist to set up my appointment.
I have genital herpes (7 years) and take Valtrex 500 mg daily to protect my partner. Currently paused on the Valtrex for now while I wait for the doctors to see me/figure out what it is.
Has anyone else experienced this before?
submitted by sprout-andthebean to HSVpositive [link] [comments]


2024.05.18 16:47 Drifter_of_Babylon PSA God told me to talk to you about why your aquarium has cloudy water.

submitted by Drifter_of_Babylon to petsmart [link] [comments]


2024.05.18 14:58 JadedBarber5363 [WTS] [WTT] 1 Gram Palladium, Slabs, 1/10 Gold Phil, Proof Sets, Ike’s, 10 oz Bars, 5 oz Bars, 1 oz Bars, 1 oz Rounds/Coins, Kooks, Geigers, Fractional, Foreign, Goldbacks, Casino Tokens, Collectables, and more.

*Chat Preferred. Only accepting trades for items under “COLLECTABLES”. Items verified using SIGMA Investor (Thru and Gravity), Pocket Pinger, Weight, and Magnet.
PROOF
GOLD
1/10 Philharmonic 1998- $255 SOLD
ROUNDS AND COINS:
7x 1 Gram Silver- $14 SOLD
2.5 Gram Silver Bar- $3
5x 1/10 Silver- $25
1/4 Oz Round- $10
1922 Peace Dollar- $26
1 oz APMEX Bar- $32
2000 The Dawn of A New Millennium 1oz Silver- $35
1 Gram Geiger In Assay- $10
40% lot ($5 Face)- $47
35% Lot (5 War Nickels)- $8.50
16x 1 oz Silver Towne Mighty Eagles- $512
4x 1 oz Buffalos- $128
7x 1 oz Misc Nicer Rounds- $230
4x Casino Tokens- $85 For All or $22 EA
5 oz Sealed Bar- $158
Cale Yarborough 1 oz Round- $45
1978 Cien Pesos- $20
4 Standing Liberty Quartahhhs- $25
1974 1 oz Liberty Medallion- $35
20x 1 oz Tokelau 2024 Proof Mustang- $36 EA for all for $34 EA
Numismatic 90% (1905 Barber Dime, 1915 Barber Quarter, 1901 O Barber Half, and 1937 Walker.)- $47
$9.50 Face 90% Lot ($6 JFK, $.50 Franklin, $1 Wash, $1.20 Rosevelt’s, $.80 Mercs all with dates and some are higher grades)- $208
5 2x 1962 Proof Sets (Some missing random pieces like cardboard or paper)- $26 EA
Blue Ike (1x 1971, 2x 1972, 3x 1974)- $13 Ea
10 oz Bar- $315
1/2 oz Round- $16 SOLD
1 oz Peace Dollar Round- $40
2x 2021 African Wildlife Elephant 1 oz- $36 EA SOLD
5x 2023 Kookaburra (W/Capsule)- $35 EA
5x 2024 Kookaburra (W/Capsule)- $35 EA
1 oz Germania Mint Allegories (W/COA)- $45
1 oz 2024 BU T2 ASE- $35
500 Gram The Holy Mint Bar- $525
6x Vintage Bars (Engelhard, JM, and Silver Towne) Lot- $215
1964 Accented Hair Proof Set (Complete)- $70
24x 2024 1/10 Silver Brittanias- $10 EA or all for $9 EA
2001 S Proof JFK Half- $12
SLABS: (Whale deal is buy all slabs get the 1900 Morgan for $60)
2022 S Dr. Sally Ride PCGS PR70 DCAM- $15 SOLD
2022 1oz Serbia Nikola Tesla Natural Healing NGC MS70- $115
1950 S Booker T Washington PCGS MS65- $65
1944 Winged Liberty Dime PCGS MS66- $40
1964 Washington Quarter NGC PF67 (Crack on slab)- $15
1946 Walking Liberty Half PCI MS65- $95
2017 S ASE NGC PF70 Ultra Cameo San Fransico Slab- $120
1922 D Peace NGC MS63- $85
1900 Morgan PCGS MS63 (Toned)- $115
1889 Morgan NGC MS63- $70 SOLD
1883 Morgan NGC “Fattie” MS64 (Toned)- $125
2023 Morgan CAC First Delivery MS69 Slab- $100
Bradford Exchange 2023 T2 BU ASE- $40
1855 Germany 1 S Hamburg MS63 NGC (Toner)- $80
RANDOMS:
5x 1 Gram Argor Palladium- $65 EA
25 2023 New Hampshire Goldback- $110 SOLD
Silver 1975 Philippines Proof Set (complete)- $90
10 Oz “The Wedge” DHF Silver Bar- $380
COLLECTABLES: Will trade for low premium Silver and Gold. [WTT] [WTS]
1x 2021 Mongolian HedgeHog Coins- $110 EA SOLD
2x 2021 Cameroon Crucifix Coin- $120 EA
6x Fiji 2022 Snowflake Coin W/Crystals (Sealed)- $85 EA
3x Fiji 2023 Wonders Of Nature Great Barrier Reef Coin- $145 EA
2021 Star Wars IG-11 NIUE Coin- $105
2021 Samoa Splash Of Colour (Color 🇺🇸) New York City Coin- $175
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Shipping/payment:
🚛*Shipping- is $5 for 1-8 oz (Ground Advantage), $6 for 9-12 oz (Ground Advantage) and $10 for over 12 oz (Priority Mail) both with tracking numbers. American shipping only 🇺🇸 (prices are for lower 48). I am shipping out of Oklahoma, if you are close I may be able to ship cheaper ground than priority on higher weights. Be sure to inquire if you expect that to be the case.
🧾*Payment- I accept Zelle (preferred), Venmo, cash app, and PayPal FF (least preferred and not available for low flair users). Please no notes/comments or I will refund (if you are forced to use one add an emoji or . ) Thanks!
💂 *Security- I will not give my password to anyone for any reason. I have enabled two factor authentication and have other steps to help protect myself and the buyers. I also use the same setup to take proof photos, so any deviation in that is a red flag 🚩.
submitted by JadedBarber5363 to Pmsforsale [link] [comments]


2024.05.18 12:33 Fragrant_Tutor8631 Trying Toniiq Berberine for Weight Loss: My Review and What You Might Expect

Toniiq Berberine shows some promise in regulating blood sugar levels, which can assist in controlling appetite to a degree. However, its effectiveness in weight loss is not as significant or consistent, and some users report adverse side effects like gastrointestinal discomfort.
For those seeking more reliable appetite suppression or weight management solutions, products containing a blend of fiber-rich ingredients (I recommend PhenQ, Instant Knockout, Blood Sugar Optimizer) or clinically proven weight loss agents like orlistat may offer more substantial results without the harsh side effects.

How to take Toniiq Berberine

If you're considering using Toniiq Berberine for weight loss or appetite control, here's my take based on personal experience.
I started with a lower dose berberine product called CandiBactin, which was easier on my stomach and helped avoid constipation issues that can sometimes come with berberine supplements.
After finishing the first bottle, I switched to a higher-quality berberine like Toniiq, expecting to see continued improvements without the digestive side effects.
I learned the hard way that you've got to be careful about mixing supplements. With CandiBactin, I didn't realize that taking probiotics at the same time would mess things up.
MOst people would end up feeling bloated and constipated if you mix berberine and probiotics at same time, losing all the initial benefits.
When I switched to just the Toniiq Berberine, I started taking probiotics again, which helped settle my stomach and kept the constipation at bay. It's crucial to find the right balance and timing for taking these supplements to avoid any unpleasant side effects.
Timing is everything with these kinds of supplements. I'll advise taking your berberine in the morning on an empty stomach, followed by my morning coffee (preferabely with collagen).
This would help kickstart the day without any issues.
I'd take the second dose right before dinner, not with or after, to avoid any late-day digestive issues.
If you're starting out, you might want to experiment with different times to see what works best for your body.
Remember, what works for one person might not work for another, so it’s all about finding your rhythm.
Also, If you're looking for stronger appetite suppression than berberine alone, consider adding fiber supplements like nopal cactus OR glucomannan to your regimen.
Nopal cactus and Glucomannan, in particular, expand in the stomach to create a feeling of fullness, potentially reducing your overall food intake more effectively than berberine's mechanism of interacting with brain pathways.
Additionally, you might explore weight management supplements like PhenQ (Nopal cactus) and Instant Knockout (Berberine), which combine various ingredients known for boosting metabolism and enhancing fat burning alongside appetite suppression.
JUST for the weight loss & appetite suppression, I'd rate Toniiq at around 3/5.
Introduction
Toniiq Berberine stands out in the crowded market of berberine supplements due to its exceptionally high purity levels. Marketed as being 97% pure, it claims to deliver a more potent dose compared to many other berberine products.
This high concentration might seem promising if you're looking for something that packs a stronger punch in terms of potential health benefits. However, it’s crucial to approach these claims with a healthy dose of skepticism, as the actual experience can vary widely.
The product is derived from Berberis Aristata (Indian Barberry) sourced from the mineral-rich foothills of the Himalayas, which is highlighted as a unique selling point.
The wild harvested aspect suggests it might contain more of the active components, but again, whether this translates into tangible health benefits is subject to individual differences and should be considered critically.

Pros & Cons of Toniiq Berberine as a Natural Ozempic Alternative

Pros:

Cons:

Evaluation of 500 mg Berberine HCI as a Natural Ozempic Alternative

1. Blood Sugar Regulation: 4/5 Berberine HCI is well-regarded for its ability to regulate blood sugar levels, which is crucial for mimicking some of the glucose-managing effects of Ozempic. Numerous studies suggest that berberine can help decrease insulin resistance and improve glucose uptake in the cells, making it a strong candidate for naturally stabilizing blood sugar levels.
2. Appetite Modulation: 3/5 While berberine is primarily known for its role in blood sugar management, it may also help in moderating appetite to some extent. The effect on appetite is less direct compared to Ozempic; however, by stabilizing blood sugar levels, it can reduce sudden hunger spikes and help in controlling cravings, albeit less significantly.
3. Metabolic Enhancement: 3/5 Berberine has shown potential in enhancing metabolic functions by activating AMP-activated protein kinase (AMPK), which is often referred to as a metabolic master switch. This activation can aid in improving metabolic health, although the effect might not be as pronounced as the metabolic enhancements provided by Ozempic.
4. Energy Optimization: 2/5 The impact of berberine on energy levels isn't as significant as its other benefits. While better blood sugar control can indirectly contribute to improved energy levels by preventing energy dips associated with glucose fluctuations, berberine does not inherently boost energy in the way stimulants or specific energy-enhancing supplements might. Therefore, its effectiveness as an energy optimizer is relatively modest compared to Ozempic's overall benefits to vitality and physical activity support.
5. Weight Management Support: 3/5 Berberine has shown some potential in supporting weight management, primarily through its ability to improve insulin sensitivity and metabolic function, which can help in fat reduction. However, the direct effects on weight loss are not as consistent or significant as those seen with Ozempic, where the mechanism is more directly linked to weight reduction.
6. Safety and Tolerability Profile: 4/5 Berberine is generally considered safe for most adults when taken at recommended dosages. However, it can cause some gastrointestinal side effects such as diarrhea, constipation, or abdominal discomfort, particularly when taken in high doses. Overall, it has a good safety profile, but like any supplement, it should be used under the guidance of a healthcare professional to manage potential side effects.
7. Inflammatory Response Reduction: 3/5 Berberine has anti-inflammatory properties that may help in reducing systemic inflammation. This reduction can indirectly support insulin sensitivity and metabolic health, which are beneficial in managing conditions like diabetes and obesity. The anti-inflammatory effects are helpful, but they may not be as robust or well-studied as those associated with pharmaceutical options like Ozempic.
8. Gut Health Improvement: 2/5 While there is some evidence to suggest that berberine can influence gut health by modulating gut bacteria, these effects are not as well documented or understood as its other uses. The potential benefits to gut health could contribute to improved overall well-being and metabolic health, but more research is needed to fully understand and confirm these effects, making it a less reliable option for gut health improvement compared to Ozempic's more comprehensive benefits
submitted by Fragrant_Tutor8631 to HealthTrendz [link] [comments]


2024.05.18 12:27 WhichIndependent5060 I am cured!!! There is hope after all.

I 21(M) have successfully cured my ed. Started experiencing it around 18 shortly after getting covid and it was mild and just kept gradually getting worse until the point I had no drive or will to even have sx, couldn't even get hard if i focused. No libido. I tried multivitamins/no fap/exercise/keto/blood tests and damn near everything I saw lurking on this sub to no avail.
I am NOT a quiter though so I locked in hard. Many sleepless nights of research experimenting and here I stand CURED. I think I'm even more sexual now than the heights of puberty and its honesty kinda annoying but I'm glad for it definitely better than zero libido.
What cured me? I'm not fully sure what exactly it was but here is what I did before noticing change.
Zinc - 50mg taken before a workout Magnesium - 500 mg taken before a intense workout Vitamin A - 10000iu taken with breakfast ( I think this may be the major catalyst for the change I experienced because I started getting better 1 week after taking while I had been on the zinc and mag for 2 weeks) Beetroot juice - a cup every other night when I started getting better and it has helped a lot now I'm so hard sometimes it's painful. Chewing food thoroughly at every meal - I am one to eat quickly but in my research I found that this can lead to problems in your gut as well as problems with nutrient absorption so I cut that out. Stoped sucking in the gut- Its a habit I developed to hide my tummy but in my research I saw it can cause problems like pelvic floor problems and even raise cortisol levels along with other issues.
I hope this post helps someone struggling and give you a bit of hope because things can definitely get better. Just keep swimming;)
submitted by WhichIndependent5060 to erectiledysfunction [link] [comments]


2024.05.18 12:15 Lopsided_Seat_9611 Your tips and tricks?

Yesterday it began with the visual aura (my usual first symptomps). This glittering thing going wider and wider. I was really sad that in an hour it would be two days of hell. I wen to find anything that could help. Usually sumatriptan makes the worst symptoms last for 8 hours instead of 24. But with the pain,not ability to move or do anything except suffer.
I was desperate that I took this steps: aspirin 500 mg sumatriptan 50 mg 6 minutes HIIT glass of water cola zero (trying to find coffein) cold shower and then hot shower for the neck lying on the bed with my head down and massaging it... and them I went to bed to lying down and may be sleep at least 15 minutes before this pain goes wide...
Guys, it helped. I feel numbness in the half of the head, but like only 5% of this Crucis pain and I am able to walk, talk and live!!!
I have no idea which step or combination helped. Have you got your recipes to make pain stop at the beginning?
submitted by Lopsided_Seat_9611 to migraine [link] [comments]


2024.05.18 11:48 Fragrant_Tutor8631 My Luma Nutrition Breberine review: best berberine for appetite suppression?

Quick Summary

Luma Nutrition Berberine offers a high dosage of berberine hydrochloride aimed at supporting weight loss, appetite control, and blood sugar regulation. However, user feedback reveals mixed results, with some experiencing significant benefits while others report no effect or adverse side effects such as gastrointestinal distress and dizziness.
I think overall, it's a decent berberine supplement for various berberine related health effects.
But given the variability in effectiveness and the potential for severe side effects, it appears that Luma Nutrition Berberine may be overly hyped, especially for those looking for consistent and safe results in weight management and metabolic health.
For appetite suppression & weight loss, I'd rate it at 3, I'd rather recommend something like PhenQ and Instant Knockout for appetite management & weight loss (both are better appetite suppressants than Luma), while Blood Sugar Optimizer does a better job in blood sugar management , being my top pick as (Berberine + Ceylon cinnamon supplement).
Introduction
If you've stumbled upon Berberine while searching for natural supplements to aid weight loss or control appetite, you might be curious about what it actually does.
Berberine is a compound found in several plants that is often used in traditional Chinese medicine. It’s known for its potential to impact blood sugar levels and improve metabolic health. Recently, it's also been gaining attention as a weight loss and appetite control aid and as a natural supplement that cna have some ozempic like effects..
Luma Nutrition is just one of many brands offering Berberine, but they promote their product with some pretty appealing features like being all-natural and vegan-friendly.
If you're curious about trying Luma Berberine, especially for controlling blood sugar and cholesterol, it might do some good.
Now different people have different kinds of expereinces with Berberine.
From my own year-long experience with berberine, not Luma specifically but a similar product, I found that it really helped with things I wasn't even targeting initially—like my chronic migraines pretty much vanished, and to my surprise, my thinning hair got thicker. It also worked wonders for my sleep; I can pretty much clock out the minute I hit the pillow and get a solid 7.5 hours without a hitch. But don't expect miracles in weight loss—I didn't shed pounds, and frankly, it didn't make a dent in my appetite at all.
Now, it's important to talk about how you might feel when starting out and as you continue.
Early days, you might notice some mild stomach issues like gas because your body's getting used to it.
Berberine is tough to absorb, so take it on an empty stomach, about 30 minutes before you eat, to get the most out of it. Luma is no exception!
As time goes on, if you don't give yourself breaks from it, you might see some downsides.
For some people , taking it continuously may lead to some sexual dysfunction and mess with gut health, so I switched to a schedule—500 mg once daily for five days a week and then a break every few weeks.
This routine keeps the side effects in check and helps maintain the benefits without overwhelming my system. So, if you're planning to try it, consider a similar approach to manage any potential issues effectively.

Pros & Cons of Luma Nutrition Berberine

Pros:

Cons:


Overview of Luma Nutrition Berberine
Luma Nutrition's Berberine comes in capsule form, which I found pretty standard for most dietary supplements. Each bottle contains 60 capsules, with a recommended dosage of two capsules per day.
This means each bottle is essentially a one-month supply if you follow the recommended dosage. The capsules themselves are a bit on the larger side, which might be a concern if you have trouble swallowing pills, but I managed fine.
One of the big selling points Luma Nutrition pushes is that their Berberine is made from all-natural ingredients, is vegan-friendly, and manufactured right here in the USA.
These are nice boxes to check off for anyone who's mindful about the ethical aspects of their supplements. They also claim that their product contains no GMOs, and is free from artificial fillers or dyes—something I appreciate since I try to keep my consumption of unnecessary additives to a minimum.
An independent company has verified the contents and safety of the product, which helps in building trust, especially if you're cautious about the purity of supplements. I found these details important not just for peace of mind but also because when it comes to supplements, you really want to make sure you're ingesting something safe and as advertised.

My Ratings Evaluation of Luma Nutrition Berberine as a Natural Ozempic Alternative

Blood Sugar Regulation

Rating: 4/5
Luma Nutrition Berberine contains 1200mg of Berberine HCI derived from Berberis aristata per serving, which is known for its potent effects on lowering blood sugar levels. Clinical studies have shown that Berberine can be effective in managing blood sugar, making it a strong natural alternative for mimicking some of the glucose-regulating effects of Ozempic. However, it might not be as consistently powerful as Ozempic, hence the deduction of one point.

Appetite Modulation

Rating: 3/5
While Berberine is primarily known for its impact on blood sugar and cholesterol, its effects on appetite are less direct. Some users report reduced appetite possibly due to better blood sugar control, which can decrease cravings and help with weight management. However, it's not specifically an appetite suppressant like Ozempic, so it scores a middle range for this category.

Metabolic Enhancement

Rating: 3/5
Berberine is reputed to have positive effects on metabolic health, primarily through the enhancement of glycolysis, which helps the body break down sugars more efficiently, and by improving insulin sensitivity. These actions can contribute to a higher metabolic rate indirectly. However, compared to Ozempic, which has a more direct role in enhancing metabolic outcomes, Berberine's effects are more moderate.

Energy Optimization

Rating: 2/5
While Berberine has been noted for its metabolic benefits, it is not primarily known for directly boosting energy levels. Users may experience increased energy indirectly through improved metabolic health and better blood sugar control, but it does not inherently contain energy-boosting properties like caffeine or other stimulants. Therefore, its capacity to support increased physical activity and vitality is limited and not as pronounced as the effects you might see with Ozempic.

Weight Management Support

Rating: 4/5
Berberine has shown promising results in supporting weight management through its ability to improve insulin sensitivity and enhance the breakdown of fats and sugars in the body. This can lead to reduced fat accumulation and support gradual weight loss, which aligns well with some of the weight loss mechanisms seen with Ozempic. While not as directly impactful as Ozempic, which is specifically designed for weight management in diabetic patients, Berberine holds its ground as a supportive supplement for natural weight management.

Safety and Tolerability Profile

Rating: 4/5
Berberine is generally considered safe when used appropriately, with common side effects being gastrointestinal discomfort such as cramps, diarrhea, or constipation. These side effects are typically mild and can often be managed with dosage adjustments. It is well-tolerated by most individuals, making it a viable alternative for those looking for natural supplements with minimal adverse effects. However, like any active compound, it's important to consult with a healthcare provider, especially for those with existing health conditions or those taking other medication.

Inflammatory Response Reduction

Rating: 3/5
Berberine has demonstrated some anti-inflammatory properties, which can contribute to overall health improvements, including enhancing insulin sensitivity and metabolic function. These benefits are crucial for managing conditions like diabetes and obesity, which are often accompanied by chronic inflammation. However, the anti-inflammatory effects of Berberine are not as extensively documented or as potent as those of some pharmacological treatments like Ozempic, which have broader systemic impacts. Therefore, while Berberine supports inflammation reduction, its effectiveness in this area is moderate compared to more targeted treatments.

Gut Health Improvement

Rating: 2/5
Berberine's impact on gut health is less clear and direct. Some studies suggest that Berberine can alter gut flora, potentially leading to improved gut barrier function and reduced permeability, which can indirectly support nutrient absorption and overall gut health. However, these effects are not as well studied or as pronounced as its effects on blood sugar and metabolism. Therefore, while there may be some potential benefits to gut health, they are not a primary function of Berberine, making it less effective in this category compared to Ozempic, which can have more noticeable effects on metabolic health and weight management through gut-related mechanisms.
submitted by Fragrant_Tutor8631 to HealthTrendz [link] [comments]


2024.05.18 06:42 Throwaway_inSC_79 I feel like a horrible dog owner. Accidentally gave my boy Naproxen

Title. I’m sick to my stomach.
He’s 71lbs, and he just got out of Sucralfate yesterday for nausea. It’s once every 12 hours. I got home from work, and instead of me taking my Naproxen, I gave it to my boy. 500 mg.
We rushed to the emergency vet and they are keeping him for the 3 days. We can call periodically for updates. I even saw him when we left. His spirits seem fine. Happy, excited.
But I can’t believe I did this to him. I’m praying for a good outcome. It was only 1 pill, given at 10:15pm. And just got home from leaving him at the vet a little after midnight.
I just feel horrible. How could I do this to him? How could I poison him?
I know it’s going to be tough. I’d just like some reassurance it’s going to be okay. Even the vet seemed hopeful, so that’s good.
Update 1: as a wake up and debate calling them (they said to call around noon but I’m worried), I didn’t have to debate long, because they called me. My heart sunk, only to hear that he’s doing good. Perky. Taking the fluids and took activated charcoal so his stool will likely be darker, but they’re going to monitor him the rest of the day. I don’t want to ask about visiting; I want them to focus on him and rather he just relax as best he could. But that was great news to hear upon waking up.
Update 2: we called them, because my mother wanted to talk to them as well. He’s doing well, peeing fine, not really eating but we told them he’s a picky eater and doesn’t like to eat in public. But they said the bloodwork they took last night provides a baseline, and they’ll know more around midnight tonight. So here’s to hoping it remains at good levels. The dr this morning made me feel hopeful, and talking now around noon, I feel less hopeful. I hope it’s just my pessimism.
submitted by Throwaway_inSC_79 to AskVet [link] [comments]


2024.05.18 06:26 FloridaMan79 Magnepan with Rotel on a Budget

Magnepan with Rotel on a Budget
Moving to a new house and figured why not get a real sound system for it. Threw some offers in FB MP, luckily a seller accepted $250 for M&L Source speakers with dynamo 500 sub. so next day I started looking for proper amp for it. Luckily someone had just listed a Magnepan complete surround speakers mg 12, mg mc1, mg cc2 for $450 and a Rotel rmb 1095 for $325. Stopped by the sellers house and he told me he's the original owner and seemed to know his gear very well and just really downsizing. So a quick eBay search with the Rotel I decided to take the 1095 and Magnepans as well. He gave me his speaker wires/power strips /manuals and even his diy amp rack. Coming from Polk tower speakers 5.1 setup this 2 speakers I bought is my first real speakers. How good is the Magnepans surround system for movies compared to newer systems? I listen to mostly rock/alt/rap/electronic bass heavy music mostly digital/flac. Is the Magnepans worth it paired with SVS SB 2000. Or stick with the ML Source? Have a newer Marantz receiver that I'm currently using and have a lot of research to do on using it with an amp. Any tips on setup with these especially how to arrange them so it won't look like a sore thumb in the living room with the spouse.
submitted by FloridaMan79 to BudgetAudiophile [link] [comments]


2024.05.18 05:46 Western-Coyote5675 I asked ChatGPT for supplements ranked by effectivenes with benefits, daily dosage and interactions with Strattera/Atomoxetine. Would appreciate your thoughts or personal experience with any of these?

Supplements for inattentive ADHD
Omega-3 Fatty Acids (EPA and DHA) - Benefits: Supports brain function and may improve cognitive symptoms of ADHD. - Dosage: 1,000 to 2,000 mg daily.
Panax Ginseng - Benefits: Enhances cognitive function and energy levels, supports neurotransmitter activity. - Dosage: 200 mg to 400 mg of standardized extract daily.
Zinc - Benefits: Plays a role in neurotransmitter function and can help improve ADHD symptoms. - Dosage: 15 to 30 mg daily with food.
Magnesium - Benefits: Has calming effects that can benefit hyperactivity and impulsivity. - Dosage: 200 to 400 mg daily in divided doses.
Iron - Benefits: Important for neurotransmitter function; consult a healthcare provider for testing before supplementation. - Dosage: 20 to 65 mg daily, based on confirmed deficiency.
Vitamin D - Benefits: Essential for brain health; addresses common deficiencies. - Dosage: 1,000 to 4,000 IU daily.
L-Tyrosine - Benefits: A precursor to dopamine, potentially aiding in dopamine receptor recovery. - Dosage: 500 to 2,000 mg daily in the morning.
B-Complex Vitamins - Benefits: Vital for energy and brain function. - Dosage: Follow the manufacturer’s recommended dosage.
Ashwagandha - Benefits: Reduces stress and anxiety, supports overall mental well-being. - Dosage: 300 to 500 mg of root extract per day, taken in divided doses.
Acetyl-L-Carnitine - Benefits: Can improve alertness and mitochondrial function in the brain. - Dosage: 500 to 2,000 mg per day in divided doses.
Mucuna Pruriens - Benefits: Contains natural L-Dopa, supports dopamine production. - Dosage: 100 to 500 mg daily.
Ginkgo Biloba - Benefits: Helps with focus and memory. - Dosage: 120 to 240 mg daily in divided doses.
Supplement interactions with Atomoxetine * Panax Ginseng: This supplement can affect the way the body metabolizes certain medications due to its effects on enzyme activity. Panax Ginseng could potentially alter the metabolism of Atomoxetine, affecting its efficacy or side effects. Monitoring by a healthcare provider is recommended. * Magnesium: While generally safe, magnesium can sometimes have a mild inhibitory effect on the central nervous system, which might potentiate some effects of Atomoxetine, particularly if it causes sedation. This combination is typically safe but should be monitored. * L-Tyrosine: This is a precursor to dopamine, and because Atomoxetine increases the level of neurotransmitters in the brain, adding L-Tyrosine could alter the effects. There is a theoretical risk of increased side effects like jitteriness or anxiety, especially at high doses of L-Tyrosine. * Ginkgo Biloba: Ginkgo can influence blood clotting and might increase the risk of bleeding. Although this is generally more of a concern when taken with anticoagulants, caution is advised when combining it with any other medication, including Atomoxetine, particularly if there are any known bleeding risks.
submitted by Western-Coyote5675 to StratteraRx [link] [comments]


2024.05.18 05:44 I_read_reddits_rules You're in a show like Naked and Afraid, but in this case, if you win, you get $100 000. You get to choose one of these options of things to help you. What do you choose? (click for details)

It's you and 8 others. The only things you are given as a group is a 2-gallon pot and a machete.
Each of you rolled dice and you won—you won the choice of any of these 6 options.
No one's allowed to take what you'd choose from you without your permission—you can use, lend, give away, and/or bargain with such as you wish. If anyone steals from you wt:thon would be kicked out by the show's producer(s).
The area is not quite a jungle, nor a desert, but somewhat in-between.
The game is for 3 months. If you make to then, or be the last standing if none of you do (minimally after a month), you get $100 000.

option 6:
10 ℓ of sunscreen, a 1 - 15 year-old bottle of aspirin (500 - 500 mg tablets), a 60 x 60 x 20 cm block of ice, a charged electric drill (no bits), and 1 pink and yellow skirt with a 8 cm length and 75 cm waist.
View Poll
submitted by I_read_reddits_rules to pollgames [link] [comments]


2024.05.18 05:43 TheTruthHurtsBabes Allergic to Acetaminophen?

Now before I start, I know this makes me sound like a drug seeker. But i’m a 21 year old healthy female. I don’t do drugs (yes that includes weed, and I haven’t drank in months). I’ve noticed now for years that anytime I take acetaminophen, I feel like i’m buzzing. My chest and neck near my carotid arteries get tight as well as my throat. My heart races and stays high for 12-24 hours after taking it. I get all hot and impending doom. I’m not sure if i’m allergic since I don’t swell or get a visible rash. I thought it was normal to feel this way until yesterday at work when I took 500 mg fast acting off brand tylenol for a headache. I literally felt like I couldn’t breathe and needed to step out for a second for air (didn’t help). The rest of the night and this morning I felt tingly and my head felt weird. The chest tightness and neck/throat tightness went away after a few hours though. There was no caffeine in the active or inactive ingredients. I can’t explain this buzzing feeling other than I feel like i’m dying and it drives me insane:/
submitted by TheTruthHurtsBabes to Allergies [link] [comments]


2024.05.18 04:52 bhamidipatiSK Why the discrepancy between BD FACSAria III Fusion and hemocytometer nuclei counts?

Hello!
I'm working on standardizing a protocol for a new snRNA-seq platform we're testing. For this, I'm doing FANS to sort nuclei that I can input into this platform. I've been working on this for a while, but the biggest unresolved problems are the nuclei count numbers and integrity. I have some questions and concerns below that I'd really appreciate any suggestions/recommendations about.
At the end of this post, I've included the following in brief:
Problems
  1. Nuclei count discrepancy:
    • The sorted nuclei numbers that BD FACSDiva 8.0.2 gives me are an over-estimate by a wide margin compared to what I get when I count them manually with a hemocytometer. For example, in the most recent run, the counts according to BD Aria III for the three populations I was sorting were:
      • NeuN+GFP+: 10,500
      • NeuN+GFP-: 50,000
      • NeuN-GFP-: 50,000
    • BUT, the hemocytometer counts (counted after mixing 1:1 with Trypan Blue) were:
      • NeuN+GFP+: 3,300
      • NeuN+GFP-: 12,600
      • NeuN-GFP-: 8,400
  2. Collection volume:
    • Right now, the final collection volume is around 60µL. I want to be able to collect the nuclei in a small volume (~5 µL total) because that's what the sequencing protocol recommends. I know I can spin it down, but I'm worried that spinning it down and reconstituting would lead to further nuclei loss.
Questions and concerns:
  1. Why is there a large discrepancy between the BD FACSDiva and hemocytometer counts?
  2. What are the best practices to minimize nuclei loss and maintain integrity, especially when handling small volumes?
  3. Are there specific protocols or tips for accurately counting fragile nuclei? I have tried doing an AO/PI stain (Logos) and counting using Countess FL, but the numbers are poor, consistent with hemocytometer counts.
  4. How can I ensure the sorted populations are as pure and intact as possible?
Background
Experiment design PV-Cre mouse crossed with a nuclear GFP reporter line such that Cre+ cells express nuclear GFP. I want to sort nuclei from three populations: PV neurons (NeuN+GFP+), non-PV neurons(NeuN+GFP-), and non-neurons (NeuN-GFP-).
Nuclei isolation I isolated nuclei from frozen mouse cortical tissue using an in-house nuclei isolation protocol (below). Before sorting, I incubated the nuclei suspension with 2% BSA for 10 minutes, followed by a 10-minute incubation with Anti-GFP (FITC-conjugated), Anti-NeuN (Alexa Fluor 647-conjugated) antibodies, and 1 mg/ml DAPI.
Nuclei isolation protocol The protocol involved transferring frozen brain tissues to pre-chilled Dounce homogenizers containing 1 ml of NIM buffer (containing sucrose, KCl, MgCl₂, Tris-HCl (pH 7.4), DTT, protease inhibitor, RNase inhibitor, Triton X-100). The tissues were gently homogenized on ice with ice-cold pestles for 10-15 strokes. The homogenate was transferred to pre-chilled microcentrifuge tubes and centrifuged to pellet the nuclei. After aspirating the supernatant, the pellet was gently resuspended in 1 ml of ice-cold NIM buffer and centrifuged again at 1000 g for 8 minutes at 4°C. The final pellet was resuspended in 450 µl of NSB nuclei storage buffer (sucrose, MgCl₂, Tris-HCl (pH 7.4), DTT, protease inhibitor, RNase inhibitor), filtered through a 40 µm cell strainer, and incubated with nuclease-free BSA to prevent clumping. The suspension was then incubated with the antibodies listed above.
Fluorescence-Activated Nuclei Sorting (FANS) FANS of single nuclei was performed using the BD FACSAria III Fusion with a 70 µm custom nozzle at a drop-drive frequency of 87.2 kHz, sample pressure: 52 psi, Cytometer Setup and Tracking (CST) enabled, and the laser and detector configuration was 2B-2R-4V-3YG-2UV.
Gating strategy
Laser and filter settings
Drop delay
Thank you!
submitted by bhamidipatiSK to flowcytometry [link] [comments]


2024.05.18 04:51 bhamidipatiSK Why the discrepancy between BD FACSAria III Fusion and hemocytometer nuclei counts?

Hello!
I'm working on standardizing a protocol for a new snRNA-seq platform we're testing. For this, I'm doing FANS to sort nuclei that I can input into this platform. I've been working on this for a while, but the biggest unresolved problems are the nuclei count numbers and integrity. I have some questions and concerns below that I'd really appreciate any suggestions/recommendations about.
At the end of this post, I've included the following in brief:
Problems
  1. Nuclei count discrepancy:
    • The sorted nuclei numbers that BD FACSDiva 8.0.2 gives me are an over-estimate by a wide margin compared to what I get when I count them manually with a hemocytometer. For example, in the most recent run, the counts according to BD Aria III for the three populations I was sorting were:
      • NeuN+GFP+: 10,500
      • NeuN+GFP-: 50,000
      • NeuN-GFP-: 50,000
    • BUT, the hemocytometer counts (counted after mixing 1:1 with Trypan Blue) were:
      • NeuN+GFP+: 3,300
      • NeuN+GFP-: 12,600
      • NeuN-GFP-: 8,400
  2. Collection volume:
    • Right now, the final collection volume is around 60µL. I want to be able to collect the nuclei in a small volume (~5 µL total) because that's what the sequencing protocol recommends. I know I can spin it down, but I'm worried that spinning it down and reconstituting would lead to further nuclei loss.
Questions and concerns:
  1. Why is there a large discrepancy between the BD FACSDiva and hemocytometer counts?
  2. What are the best practices to minimize nuclei loss and maintain integrity, especially when handling small volumes?
  3. Are there specific protocols or tips for accurately counting fragile nuclei? I have tried doing an AO/PI stain (Logos) and counting using Countess FL, but the numbers are poor, consistent with hemocytometer counts.
  4. How can I ensure the sorted populations are as pure and intact as possible?
Background
Experiment design PV-Cre mouse crossed with a nuclear GFP reporter line such that Cre+ cells express nuclear GFP. I want to sort nuclei from three populations: PV neurons (NeuN+GFP+), non-PV neurons(NeuN+GFP-), and non-neurons (NeuN-GFP-).
Nuclei isolation I isolated nuclei from frozen mouse cortical tissue using an in-house nuclei isolation protocol (below). Before sorting, I incubated the nuclei suspension with 2% BSA for 10 minutes, followed by a 10-minute incubation with Anti-GFP (FITC-conjugated), Anti-NeuN (Alexa Fluor 647-conjugated) antibodies, and 1 mg/ml DAPI.
Nuclei isolation protocol The protocol involved transferring frozen brain tissues to pre-chilled Dounce homogenizers containing 1 ml of NIM buffer (containing sucrose, KCl, MgCl₂, Tris-HCl (pH 7.4), DTT, protease inhibitor, RNase inhibitor, Triton X-100). The tissues were gently homogenized on ice with ice-cold pestles for 10-15 strokes. The homogenate was transferred to pre-chilled microcentrifuge tubes and centrifuged to pellet the nuclei. After aspirating the supernatant, the pellet was gently resuspended in 1 ml of ice-cold NIM buffer and centrifuged again at 1000 g for 8 minutes at 4°C. The final pellet was resuspended in 450 µl of NSB nuclei storage buffer (sucrose, MgCl₂, Tris-HCl (pH 7.4), DTT, protease inhibitor, RNase inhibitor), filtered through a 40 µm cell strainer, and incubated with nuclease-free BSA to prevent clumping. The suspension was then incubated with the antibodies listed above.
Fluorescence-Activated Nuclei Sorting (FANS) FANS of single nuclei was performed using the BD FACSAria III Fusion with a 70 µm custom nozzle at a drop-drive frequency of 87.2 kHz, sample pressure: 52 psi, Cytometer Setup and Tracking (CST) enabled, and the laser and detector configuration was 2B-2R-4V-3YG-2UV.
Gating strategy
Laser and filter settings
Drop delay
Thank you!
submitted by bhamidipatiSK to molecularbiology [link] [comments]


2024.05.18 04:40 bhamidipatiSK Discrepancy between BD FACSAria III Fusion and hemocytometer nuclei counts

Hi labrats!
I'm working on standardizing a protocol for a new snRNA-seq platform we're testing. For this, I'm doing FANS to sort nuclei that I can input into this platform. I've been working on this for a while, but the biggest unresolved problems are the nuclei count numbers and integrity. I have some questions and concerns below that I'd really appreciate any suggestions/recommendations about.
At the end of this post, I've included the following in brief:
Problems
  1. Nuclei count discrepancy:
    • The sorted nuclei numbers that BD FACSDiva 8.0.2 gives me are an over-estimate by a wide margin compared to what I get when I count them manually with a hemocytometer. For example, in the most recent run, the counts according to BD Aria III for the three populations I was sorting were:
      • NeuN+GFP+: 10,500
      • NeuN+GFP-: 50,000
      • NeuN-GFP-: 50,000
    • BUT, the hemocytometer counts (counted after mixing 1:1 with Trypan Blue) were:
      • NeuN+GFP+: 3,300
      • NeuN+GFP-: 12,600
      • NeuN-GFP-: 8,400
  2. Collection volume:
    • Right now, the final collection volume is around 60µL. I want to be able to collect the nuclei in a small volume (~5 µL total) because that's what the sequencing protocol recommends. I know I can spin it down, but I'm worried that spinning it down and reconstituting would lead to further nuclei loss.
Questions and concerns:
  1. Why is there a large discrepancy between the BD FACSDiva and hemocytometer counts?
  2. What are the best practices to minimize nuclei loss and maintain integrity, especially when handling small volumes?
  3. Are there specific protocols or tips for accurately counting fragile nuclei? I have tried doing an AO/PI stain (Logos) and counting using Countess FL, but the numbers are poor, consistent with hemocytometer counts.
  4. How can I ensure the sorted populations are as pure and intact as possible?
Background
Experiment design PV-Cre mouse crossed with a nuclear GFP reporter line such that Cre+ cells express nuclear GFP. I want to sort nuclei from three populations: PV neurons (NeuN+GFP+), non-PV neurons(NeuN+GFP-), and non-neurons (NeuN-GFP-).
Nuclei isolation I isolated nuclei from frozen mouse cortical tissue using an in-house nuclei isolation protocol (below). Before sorting, I incubated the nuclei suspension with 2% BSA for 10 minutes, followed by a 10-minute incubation with Anti-GFP (FITC-conjugated), Anti-NeuN (Alexa Fluor 647-conjugated) antibodies, and 1 mg/ml DAPI.
Nuclei isolation protocol The protocol involved transferring frozen brain tissues to pre-chilled Dounce homogenizers containing 1 ml of NIM buffer (containing sucrose, KCl, MgCl₂, Tris-HCl (pH 7.4), DTT, protease inhibitor, RNase inhibitor, Triton X-100). The tissues were gently homogenized on ice with ice-cold pestles for 10-15 strokes. The homogenate was transferred to pre-chilled microcentrifuge tubes and centrifuged to pellet the nuclei. After aspirating the supernatant, the pellet was gently resuspended in 1 ml of ice-cold NIM buffer and centrifuged again at 1000 g for 8 minutes at 4°C. The final pellet was resuspended in 450 µl of NSB nuclei storage buffer (sucrose, MgCl₂, Tris-HCl (pH 7.4), DTT, protease inhibitor, RNase inhibitor), filtered through a 40 µm cell strainer, and incubated with nuclease-free BSA to prevent clumping. The suspension was then incubated with the antibodies listed above.
Fluorescence-Activated Nuclei Sorting (FANS) FANS of single nuclei was performed using the BD FACSAria III Fusion with a 70 µm custom nozzle at a drop-drive frequency of 87.2 kHz, sample pressure: 52 psi, Cytometer Setup and Tracking (CST) enabled, and the laser and detector configuration was 2B-2R-4V-3YG-2UV.
Gating strategy - Initial gating on forward scatter area (FSC-A) and side scatter area (SSC-A) to exclude debris. - Doublets were excluded using FSC-A vs. FSC-W. - Live cells were further gated on SSC-A vs. BV421-A. - NeuN+ and NeuN- populations were identified based on Alexa Fluor 647-A fluorescence. - GFP+ and GFP- populations were determined based on FITC-A fluorescence.
Laser and filter settings - FITC: 488 nm laser, 530/30 filter - Alexa Fluor 647: 640 nm laser, 670/30 filter - BV421: 405 nm laser, 450/50 filter
Drop delay - Drop Delay: 70 µm - Amplitude: 2.3 - Frequency: 87.2 kHz - Drop 1: 197 - Gap: 7
Thank you!
submitted by bhamidipatiSK to labrats [link] [comments]


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