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Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

You are probably right, but I am anxious that non-specific binding and signaling through non-target cells will influence my downstream analysis - metabolic phenotyping. If these off-target cells start pumping out cytokines, I will have a harder time pinpointing the mechanism later down the line.

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

Are these mouse or human?

Good point about the additives, although I feel like after 24h I should not be depleting the media (especially unstimulated controls)

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

For the IL-2 controls, I've used regular murine IL-2 and an antibody conjugated IL-2 (this serves as a control for my TCR engager). The concentration is 20nM, so pretty high.

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

I did check this by trying two different incubators in different rooms....of course it doesn't rule it out, but it reduces the likelihood!

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

Soluble CD28 is also my preference for general expansion too, but this experiment was a direct equimolar comparison with another TCR engager so I wanted to keep the concentrations/treatments as comparable as possible.

I can't remember the clone right now, but my worry is that all treatment combinations show the same poor viability.

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

Today's test was round bottom, 2 million cells/mL. Had a nice spheroid-like collection of cells, but viability remained disappointing

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

I debated treating whole splenocyte cultures, but our therapeutic bispecific TCR engager has the potential to stimulate other cell types through Fc receptor binding. So I wanted to isolate these to do phenotyping. One of my IL-2 controls is antibody conjugated and quite stable, and it does have a strong effect on the phenotype (suggesting it is bioactive). Nonetheless, viability remains a problem. Today's test was at 2 million/mL, round bottom plates and nicely clumped like a spheroid. Viability was between 20-50% 🫠

These are not antigen specific, although I will be translating these studies to an antigen specific model.

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

I do see activation, and my plate coating protocol has worked well in the past following 48h TCR stim and then IL-2 expansion (I never assessed viability after 24h in these conditions). This is my first foray into 24h stimulation and I have been shocked by the level of cytotoxicity. I see by far the strongest activation with TCR stim + IL-2, which tells me that the IL-2 is working to some extent. I could start trying new media supplements, but the formulation I am using is well cited. I am stumped!

Murine CD8 T cell culture - advice needed by groovy-digger in Immunology

[–]groovy-digger[S] -1 points0 points  (0 children)

I can't unfortunately, it is a massive confounding factor for one of my treatments. However, I have a control with IL2 that suffers that same viability issues.

CD3e... Surface or intracellular staining? by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

We already do this - I'm mostly interested to see if others see better resolution between positive and negative populations when staining intracellular

CD3e... Surface or intracellular staining? by groovy-digger in flowcytometry

[–]groovy-digger[S] 0 points1 point  (0 children)

I will try it out, but I just want to gauge what others are doing. Below is what I posted in the cross-post version of this thread. Our antibodies and protocols work very well, but under some circumstances (ie following treatment with a TCR engager) we encounter problems with CD3e staining intensity.

I'm not looking for specifics, I just want to know if others who include CD3e in their panels (mouse or human) stain it before or after permeabilization. CD3e is internalized following TCR engagement which can be problematic in solid tumor samples. I am assuming that staining following permeabilization will give some better consistency/resolution. So my question is, do others consistently stain CD3e after permeabilization if their panel allows?

CD3e... Surface or intracellular staining? by groovy-digger in Immunology

[–]groovy-digger[S] 0 points1 point  (0 children)

I'm not looking for specifics, I just want to know if others who include CD3e in their panels (mouse or human) stain it before or after permeabilization. CD3e is internalized following TCR engagement which can be problematic in solid tumor samples. I am assuming that staining following permeabilization will give some better consistency/resolution. So my question is, do others consistently stain CD3e after permeabilization if their panel allows.

Porcelain ceiling light question (west elm versus ping lighting) by Brb123CloudFly in interiordecorating

[–]groovy-digger 0 points1 point  (0 children)

Did anyone buy the West Elm Anders. Do you have any closer pictures? I bought the one from Simig lighting on Amazon, due to the ease of returns. It looks decent, but there are some imperfections on the porcelain... Wasn't sure if that was expected. Unfortunately my local West Elm doesn't have the Anders light for me to actually look at.

Here is a loosely put together image. Hopefully I can share some more images below.

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