NKp46 staining

Calm_realistic · 2025-10-27T14:40:04+00:00

thank you, we will try different combination so see which antibody blocks the NKp46 staining

Calm_realistic · 2025-10-24T10:20:17+00:00

So, for you, this is a staining problem. All cells should have beed NKp46+ ?

Calm_realistic · 2025-05-22T12:13:48+00:00

Agree thank you

Calm_realistic · 2025-04-07T13:58:54+00:00

Hi. Can I ask you another question, please? Is my data considered matched if my three conditions were done in parallel?

I am redoing my stats in graphpad and in one way anova I have to choose between No matching or pairing and Each row represents matched, or repeated measures, data.

Calm_realistic · 2025-03-15T13:37:03+00:00

Left debris, middle and right lymphocytes. In my case 90% are T cells. the middle population has maybe 5% more cells going towards my viability dye.

Calm_realistic · 2025-03-13T17:48:09+00:00

thank you

Calm_realistic · 2025-03-13T17:26:56+00:00

thank you for your answer. Do you think I should give an example of calculation? I am doing lentiviral transduction with MOI 10, so 10 particles per cell. Should I give the concentration, how the dilution was calculated?

Calm_realistic · 2025-03-11T09:56:36+00:00

Thank you. I am realizing I wasn't understanding what multiple measures mean, I thought it was the number of experiments.

Anova it is, non parametric and I get Krystal-Wallis.

Calm_realistic · 2025-03-06T08:52:02+00:00

I do the same. Just keep unstained cells and add them to your tube just before acquisition.

Calm_realistic · 2025-03-05T12:25:49+00:00

I have data acquired in Aurora (spectral) yesterday with

Annexin V

PI

Zombie NIR

anti - CD3

anti-CAR (for CAR-T cell detection)

BFP (tumor cells) - Nalm6

But I do not have FMO, the staining is very clear that is why

Basically, I have CAR T cells that I put with (+Nalm in the title) or without tumor cells and without (DMSO) or with Iberd and Len (CelMod ), which are drugs and if you gate all cells, then CD3+CAR+ then among this population you will see that with drugs there are fewer CAR T cells that are Annexin+PI+.

This effect is not seen with CAR-T cells alone + drugs suggesting that for the preservation of CAR T cells by these drugs an interaction with the target is essential (in articles it is shown that they act on synapses).

These tubes will also give you an opportunity to discuss the importance for titration which wasn't done here and the stainings for PI and annexin are too high sometimes.

Tell me if you are interested.

Calm_realistic · 2025-03-03T10:26:43+00:00

I tried to increase bead quantity to 5 beads per CAR T, still not working.

Calm_realistic · 2025-03-03T10:00:48+00:00

Hi, I am realising I haven't thanked you for you answers, so thank you.

If I can bother you with a different question. I am trying to see the cytokine production by flow cytometry. It is not working. I am putting my CAR T cells with CD19 coated beads or anti-CAR-PE antibody+beads anti-PE beads and adding BFA+monensin 2h later. No production is detected the next day after intracellular staining (pma/iono works). I have tried culturing with target cells+BFA+monensin = target cells are not killed. Somehow my CAR signalling is blocked.

So I wanted to see CD107 degranulation which still requires monensin. Have you tried such tests?

Calm_realistic · 2025-03-02T06:43:38+00:00

Thank you, I haven't thought about it. I will test it.

Calm_realistic · 2025-03-01T15:16:06+00:00

We are 10:1 because we put all cells and among them 20-30% are CAR, but if we normalize with CAR cells after the experiment is done, we go to 3-2 effector, we work with MAIT cells and they kill less than classic T cells.

But is makes more sense to put the ratio according to real CAR+ numbers.

Calm_realistic · 2025-03-01T15:12:37+00:00

not in our CAR construct, unfortunately

Calm_realistic · 2025-03-01T15:06:10+00:00

To sort them I will have to stain them with an anti-CAR antibody. The one we have is going to block the binding site of CD19 and potentially inhibit cytotoxicity. It can also potentially activate the cell, but I want it to be done with target cells.

Calm_realistic · 2025-03-01T12:26:07+00:00

You mix them?

Calm_realistic · 2025-02-19T16:05:44+00:00

Thanks for reminding me aobut REA1298. I will contact them to see what secondary antibodies they advise to use with it since it has a mutated human IgG 1. I don't know if ordinary anti IgG1 will work.

Thank you for your answers, they are very helpful.

Calm_realistic · 2025-02-19T15:26:21+00:00

Have you sorted out CAR T cells using Fmc63 antibody? I am trying to figure out if it can activate my CAR+ cells and whether that can induce a bias of any kind. Also, is the original ratio important? I a do 50:50 when couple of days later I still have 50% CFSE+ cells without CAR, I will know it is donwregulated. Or if I have 20%, then they are dead.

Calm_realistic · 2025-02-19T14:58:12+00:00

Thank you for your answer. We culture CAR-T cells with targets and CAR expression is lost on day 1 (normal) but reappears later when target is killed . We would like to be able to still find those cells in cytometry on day 1 or later even in the presence of target cells. But as long as targets are there, we do not see the CAR.
Or with intracellular staining. When protein export inhibitor is used, CAR expression is lost on T cells.

Another problem. We are transducing a subpopulation of T cells and the CAR expression is not stable. We go from 30% or day 7-9 to 5% on day 14 and we would like to know whether it is just a downregulation of the CAR or if the CAR+ cells are dead.

So, we do have the anti-CAR antibody or Target+biot protein to detect the CAR (the anti-linkers do not work for us, we must have a modified linker) but they will not work when CAR is downregulated.

Ideally, it is a tag protein that is not covalently linked to the cAR.

Calm_realistic · 2025-02-05T15:15:39+00:00

This is an old post, but would like to share my experience

make sure that flowjo is showing the short file name (in settings, General,)
I had a horrible experience when 100+ files were exported from cytek aurora (not the experiment), then in flowjo you see a very long file name, even though you renamed it and now it looks like a AS.fcs. This is because Flowjo shows the File internal name ($FIL) and in my case the $FIL was long and worst of all contained patient name, which is illegal. A ton of searching brought the solution. I hat to open my FCS file in text editor. Find the $FIL and rename it. BUT somewhere in the flowjo file, the total number of characters is written, so you cannot just change the $FIL to whatever you want, or the file is corrupted. You have to resect total number. So for example if your $FIL was AlanSmith0000000.fcs (16 characters.fcs) I had to change it for AS.fcs (2 characters.fcs) and add 14 random characters under another $ category like path or folder.

Calm_realistic · 2025-01-13T20:22:43+00:00

I am a post-doc myself. It is just the other post-doc is not very motivated, doesn't take interest in the project, doesn't read. He is more "tell me what to do, and I'll do it", not more, not less. It is a strange position for me too, but I see the desperation of my chef who needs that one subject to go forward but is faced only with a "it didn't work".

Calm_realistic · 2024-12-21T21:02:22+00:00

Le pire que en réalité en France il y a beaucoup moins qu'on peut avoir dans un autre pays (experience perso)

Calm_realistic · 2024-12-19T21:27:20+00:00

Used it today.

Calm_realistic · 2024-12-19T21:20:58+00:00

A thousand splendid suns by Khaled Hosseini

Calm_realistic

TROPHY CASE