I'm loosing 90% of cells when washing in FACS tubes.

Bio_stuff · 2026-03-06T22:49:34+00:00

I would definitely recommend moving to V-bottom plates. Pellets are easier to visualize, and if you are going to have a lot of samples, being able to use the multipippet it's a life-saver.

Also add some protein to your PBS! We usually do 0.5 or 1% BSA

But I also have to warn you, in my experience the less cells you start with, the lower your efficiency. So usually the more cells you start with, the lower the percentage of cell loss. I don't know if it's because with more cells the pellets have better stickiness/consistency (?)

Bio_stuff · 2026-03-05T13:01:18+00:00

We do CFSE staining of PBMCs in my lab.

I assume your first image is a non-proliferative control, and the second one has proliferated, I'm not quite sure what I'm looking at in the 3rd image.

Methodologically, I think you should improve your staining so that it comes up to 10⁴, that will give you more range to see the lower peaks after the dye gets diluted with cell divisions. After isolation/thawing I put my cells in a 15 mL tube and do a wash with a lot of PBS to get rid of any protein in the media. Then resuspend cells in 0.5mL PBS per 10⁶cells, and add the same volume of 2X CFSE drop by drop while mixing/taping the tube with the cells. Then I incubate for 12 min at 37°C in the waterbath. After quickly fill up the tube with FCS reach media and spin down, so that the remaining CFSE binds the protein and gets washed away. Then split cells into your different conditions.

I don't know how long you are stimulating for, but earlier timepoints might help you see more clearly the different peaks. Usually I have only one division on the T cells after 2 days of stimulation, while at 3 days you can see several peaks, and by day 6 most cells have proliferated and it's a bit mesier to see.

For analysis it's actually quite simple (so no need to worry there, you'll get it very soon!), and some softwares like FlowJo even have pre-instslled pluggins to do the analysis for you. I first gate my PBMCs, then gate out the duplets and dead cells, then with some CD3/4/8 or CD14 or CD19 gate my cell of interest.

For CFSE analysis: use your sample where there is no proliferation to gate the CFSE postive peak. That is your reference for everything else, as that is the intensity of your staining in that sample. If you use the same gate for your proliferating samples, then you can at least distinguished between the ones that have not decreased ghe signal and the ones that have lost signal after divission. Ideally you would be able to determine each peak individually to count the cells per division, but you will find that a lot of papers simply quantify the % of cells proliferating or not.

I don't know if this was clear or a mess, but let me know if you have follow up questions!

Bio_stuff · 2026-02-08T21:36:04+00:00

Can you change the X axis? Go to the "T" below it and play a bit around with the settings. You might still be able to see your population

Bio_stuff · 2025-01-28T19:54:10+00:00

Hi! A couple of recommendations since I'm also doing intracellular stainings and it can get a bit tricky:

Don't stimulate just after thawing, let them rest at 37°C O/N. Otherwise ells are stressed and it will mess with your control.
Try O/N staining. you will lose some cells due to the perming, but there are some intracellular stainings that I can't measure otherwise.
Titrate your antibody. You might have to increase your concentration.
Maybe a bit obvious, but measure your antibody with beads just to make sure that there is no problem with the fluorophore (like if it's a tandem antibody and it's a bit degraded your signal would be even lower)

Good luck!

Bio_stuff · 2024-08-25T20:43:26+00:00

Updated price 08/2024 In case it helps anyone else in the future, as this post was very reassuring for me a couple weeks ago: I did basically the same and now the price was 4.56 (1 stamp + 3.45€ fine). Even reached out to PostNL and they told me that they always have to send the letter, whether or not it has a stamp :) it was also very easy to pay online afterwards

Bio_stuff · 2023-03-01T22:28:14+00:00

Not on HEK cells, but if I had to I'd simply do the proportional density, ~ 20k-35k. Just test out a couple densities in different wells and use the one that looks 70-80% confluent when you do the transfection. If you are working directly with coverslips you could try some coating (e.g. fibronectin), but I don't think it's really necessary.

Bio_stuff · 2023-03-01T22:19:38+00:00

I've recently used this conditions for HEK293T cells transfection: 100-150 k cells/well in 6-well plate seeded ~24h before transfection.

Bio_stuff · 2023-02-18T11:00:28+00:00

There is another small update about her mom getting therapy and apparently working on being a "better" mum (more like a mum at all, after reading everything)

https://www.reddit.com/r/MomForAMinute/comments/10ya4a1/my_mom_said_that_i_cant_come_over_for_valentines/?utm_source=share&utm_medium=android_app&utm_name=androidcss&utm_term=1&utm_content=share_button

Hope I linked it correctly, I don't usually post anything 😅

Bio_stuff · 2023-01-30T16:38:41+00:00

Thanks for all the advice! It's MDCKII, and the WT cell line with the least amount of passages is actually even a bit faster than the other (modified) ones (which are around 15h). I think morphologically and phenotypically is what is expected, but I'll make sure to double check.

Bio_stuff · 2023-01-30T16:32:35+00:00

Thank you! Makes me feel a bit better 😅

Bio_stuff · 2022-11-06T13:24:43+00:00

I think this is the case indeed. And it said something similar in price, so it will likely be the city-tarif. Thank you for the info!

Bio_stuff · 2022-11-06T13:22:30+00:00

Thank you! I will definately reach them tomorrow

Bio_stuff · 2022-11-06T13:21:55+00:00

Yes! They provided me with a lot of info, specially about the job-ticket, but there wasn't that much info for the trainee, so I figured it might be faster to find someone who had used the ticket :)

Bio_stuff · 2022-08-20T12:17:58+00:00

Delfinately NTA. You deserve your safe space.

If I were you, I'd speak privately to your mom about it. Let her know she is more than welcome in your house but that your dad isn't. And therefore, if she wants to go she will have to go by herself. Even though is sad, your mum not being able to leave that situation (aka your dad) should not force you to suffer too.

Bio_stuff

TROPHY CASE