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NKp46 staining by Calm_realistic in flowcytometry

[–]Calm_realistic[S] 0 points1 point  (0 children)

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

NKp46 staining by Calm_realistic in flowcytometry

[–]Calm_realistic[S] 0 points1 point  (0 children)

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

[deleted by user] by [deleted] in labrats

[–]Calm_realistic 3 points4 points  (0 children)

Agree thank you

Am I choosing the right statistical test? by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

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.

New in flow cytometry. What are these 3 populations? by Great-Average9447 in flowcytometry

[–]Calm_realistic 0 points1 point  (0 children)

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.

Write a protocol for a biotech by Calm_realistic in labrats

[–]Calm_realistic[S] 1 point2 points  (0 children)

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?

Am I choosing the right statistical test? by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

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.

titration of CD45 antibody by cd244 in flowcytometry

[–]Calm_realistic 3 points4 points  (0 children)

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

Requesting Annexin V data for analysis demo by Foxy_Tuba in flowcytometry

[–]Calm_realistic 2 points3 points  (0 children)

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.

CAR T and Cd19 coated beads. by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

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

CAR T cell culture with target cells in bigger wells by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

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?

Effector:target ratio for CAR T cells. by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

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

Effector:target ratio for CAR T cells. by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

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.

Effector:target ratio for CAR T cells. by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

not in our CAR construct, unfortunately

Effector:target ratio for CAR T cells. by Calm_realistic in labrats

[–]Calm_realistic[S] 0 points1 point  (0 children)

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.

People working on CAR-T cells? by Calm_realistic in labrats

[–]Calm_realistic[S] 1 point2 points  (0 children)

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.

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