Hi! Could someone help me to solve this problem?
I will coat the 6-well plate with anti-CD3 first. Then, add anti-CD28 into T cell culture and remove T cell culture into 6-well plate. However, Jurkat cells always form clumps. Do I need to split the T cells before adding anti-CD28 into individual cells? What should I do? Centrifuge the cell culture?
Besides, I will then test the CD69 and CD25 expressed on activated T cells by flow cytometry. In this test, I will add anti-CD69-FITC and anti-CD25-PE to the cell culture. Do I also need to centrifuge the T cells to split them from clumps?
Thank you in advance!
Hi Yu Zhang
I briefly wanted to reach out concerning your inquiry. Firstly, Jurkat T cells clump a lot (especially when re-stimulated). To that end I would suggest to spin them and gently resuspend them by pipetting up and down to get a single cell suspension.
Follow the detailed extracellular staining protocol outlined here:
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Please stain for live/ dead cells (in PBS) and then for your extracellular antibodies in FACS buffer (i.e. PBS + 0.5% BSA + 0.1 % NaAzide (optional for storage).
For a very basic protocol please refer to:
https://medicine.yale.edu/immuno/flowcore/protocols/analysis/
I hope that helps.
All the best & good luck with your experiments,
Michael
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