01 August 2019 6 4K Report

Hey folks,

we’re recently facing some issues regarding the flowcytometric measurement of T cells. We are expanding T cells from peripheral blood and umbilical cord blood (UCB) MNC. We use standard protocols and stimulate the cells with CD3/CD28 antibodies. Every 3-4 days we count the cells using a flow cytomer (MACSQuant Analyzer, Miltenyi Biotec). We apply the “8-Color Immunphenotyping Kit” (Miltenyi) in a lyse/no wash protocol. The kit includes 7’AAD for dead cell exclusion.

When we start with the primary PBMC the FACS plots look all quite normal and as expected. However, after some days in culture a new population (see figure, orange circle) grows and becomes more and more dominant. The cells appear to be (dimm) CD3+ and (dimm) PI+. Interestingly, the population arises in UCB-MNC on day 5-7 of culture, and in PBMC it just starts from day 12-14 on. We are now wondering, what these cells are and if this population has to be excluded during dead cell exclusion. We thought about several explanations for the cell population, like early apoptotic cells, but as the population grows from day to day, that does not seem to make sense.

Any hints are highly appreciated. Thank you for you help.

Cheers,

Max

p.s.: Sorry for the bad plots, I’m not in the office at the moment and just used some screenshots and powerpoint to make up the figure. I’ll provide some new pictures as soon as possible.

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