u/PendingCommit98

Hey, I am titrating FOXP3-AF647 (Clone 206D from Biolegend) to identify CD4+ regulatory T cells. Everything went well, but I noticed that there was a weird population extending from the negative population at the higher concentrations which disappeared at the 1 in 200 dilution and beyond. Wondering what it could be as the far right population (which I suspect are the CD4+ Tregs) remains consistently visible.

*NOTE - I hover around the population I am confused about in the video using my cursor.

https://reddit.com/link/1x2qynl/video/676c0nyrmpuh1/player

Hey, I have done a lot of reading on flow cytometry and OMIPs but I have seen very different ways researchers use to decide what dilution to use for their antibodies. There does not seem to be one correct way of doing things and some methods seem very impractical or generally not realistic (such as using the same cell population genetically engineered to not express the antibody's target🤦).

Please share suggestions below on how to titrate antibodies for markers that produce smears or plots whereby it is hard to identify the positive population.

https://preview.redd.it/7rs0ngybl3th1.png?width=2044&format=png&auto=webp&s=c705f74c0249c3dffe3327b72c4f94ddbde2062d

Hey all, I titrated my antibody from Thermofisher, ROR gamma T - PE (clone AFKJS-9). However, this antibody looks rather odd and would love some advice or confirmation if this is what is expected of such an antibody. Please

Context: these are done on fixed PBMCs, fixed using the FOXP3 ebioscience thermofisher kit, stained for 16 hours in the fridge (100 microlitres stain volume), intracellular blocking step for 15 minutes on ice using a mix containing 2% rat serum and 2% mouse serum.

The order is ROR gamma T FMO, 1 in 1000, 1 in 5000, 1 in 10000, 1 in 12500, 1 in 15000, 1 in 17500, 1 in 20000 and 1 in 25000.

The concatenated plots are done on CD3+ cells and CD3 plus CD4+ cells.

Hey all, I titrated some more of my antibodies this week and noticed that there was a weird trail underneath the negative populations. Unsure what this could be and would love some input please. Below is the titration for CD103-BV605.

https://preview.redd.it/oj1kuhci4emh1.png?width=1326&format=png&auto=webp&s=1bee0bb6b728d07ae47f6612f00c325ce3d7a314

Key info:

• Ran the samples (human PBMCs) on the cytek aurora.
• Titrated the antibodies separately and every sample was also stained with CD3-BUV805 (1 in 200 dilution) so I could titrate my antibodies on CD3+ T cells.
• Performed autofluorescence extraction (have not noticed anything wrong with doing this).
• Unsure how it could be spectral unmixing issues if this is the case (I am still new to spectral flow)

Hey all, I recently did some spectral flow cytometry on some fixed, unstimulated PBMCs and wanted to see if the amount of mouse and rat sera affected the staining of my CD4+ lymphocytes, before titrating the rest of my antibodies.

Essentially, I thawed my PBMCs, washed them, rested them for an hour at 37 degrees Celsius and applied 100 microlitres of a blocking reagent to my cells for 15 minutes at room temperature in the dark before surface-staining them with 1:100 CD4-BUV395 for one hour on ice. After some washes, I fixed my cells and ran them on the cytek aurora (spectral flow cytometer).

I compared the effects of

1. no block (just FACS buffer)

2. Blocking mix containing 2% mouse sera (cat. 10410) and 2% rat sera (cat. 10710C)

3. Blocking mix - 5% mouse sera and 5% rat sera.

4. Blocking mix - 10% mouse sera and 10% rat sera.

5. Blocking mix - 15% mouse sera and 15% rat sera.

6. Blocking mix - 30% mouse sera and 30% rat sera.

7. Trustain Fc block (cat. 422301) at the recommended dilution of 1:20.

The results appear to show no noticeable differences with what I used so I am going to use 2% from now on.

I posted this in case it may help people in the future but please feel free to comment and critique my interpretations, decisions or anything else.

https://preview.redd.it/10apxkykq5lh1.png?width=2094&format=png&auto=webp&s=b30aa6e6439049af1a8d51f5905da4b03ab4b161

(I almost ran out of my trustain Fc block as I forgot to hit record🤦‍♂️ - hence the low cell count but the results seem interpretable.)

Hey, I wanted to exclude CD4+ regulatory T cells from my conventional CD4+ T cells and so I added a gate around the population I suspect to be regulatory T cells and continued gating on everything outside of the gate.

I do not recall seeing this done in the literature but wanted to hear if others thought this was acceptable and whether journals allow this. Thanks in advance.

https://preview.redd.it/i9eij24bn5lh1.png?width=340&format=png&auto=webp&s=416f40b1cf78de76ff102e2c37cd97872991fc13

Hey, a few months ago I did some conventional flow cytometry on unstimulated, unfixed PBMCs (thawed from frozen storage) to look at memory CD4+ T cells and I was able to identify a monocyte population above it on the initial SSC-A vs FSC-A plot. Although I did not confirm their identity with an antibody. I only stained for surface receptors.

Now I am doing spectral flow cytometry on unstimulated, fixed PBMCs (thawed from frozen storage) and the monocyte population is completely absent or at the very least markedly reduced.

I believe this may be due to fixating the cells using the ThermoFisher fixation kit (Catalog number 00-5523-00) or because of the extra wash steps I am doing as a result of an intracellular blocking step, intracellular staining step, fix/perm etc. However, if you all have any other ideas as to why this is happening please let me know. Many thanks in advance.

Extra note - I always centrifuge my cells at 300g for 5 minutes (4°C). However, once fixed, I centrifuge my cells at 600g for 5 minutes (4°C) and continue to do so for the remainder of the protocol.

https://preview.redd.it/552rog61k5lh1.png?width=1590&format=png&auto=webp&s=66012acd428a938b036728ad6ef1762983884435

Hello, I titrated my GATA3-RB705 antibody using healthy peripheral blood mononuclear cells (PBMCs) on the Cytek Aurora (spectral flow cytometer) and I am unsure how to interpret the GATA3+ population.

I thawed frozen human PBMCs (viability 98%), washed them, applied an Fc block, stained for CD3 for 1 hour on ice, fixated the cells, performed an intracellular blocking step using mouse sera (2%), and stained for GATA3 for 16 hours on ice. (I did wash steps between each of these steps).

The image shows the concatenated file showing the different antibody dilutions ranging from most concentrated (1:25) to most dilute (1:32,000).

** My thoughts are that every dilution that is more concentrated than 1:1000 is staining every cell non-sepcifically and that the true GATA3+ population is the trickle pattern above the huge concentrated blob on each of the separate readouts (I have highlighted this using a red rectangle for the 1:4000 dilution).

If I were to shoot blindly, I would say that the optimal dilution may fall somewhere between 1:2000 and 1: 8000, but before I commit to using my grant money I want to ensure I am not missing something**

Any advice or ideas is to what is going on is greatly appreciated and please let me know if you need any more details about my protocol, techniques etc.

https://preview.redd.it/2r8pi9ia4pkh1.png?width=1526&format=png&auto=webp&s=99367314eae370b12fd8180b62159830cb962138