We have published data with our collaborators on human and mouse CD4 and CD8 T cells, monocytes, NK cells, as well as dendritic cells. In theory, IsoCode chips can be used to profile any cytokine-secreting mouse or human single cell suspension.
Why do the cells need to be stimulated before loading onto the IsoCode chip?
IsoPlexis' platform uses single cell secretion profiling to determine the functional capacity of single cells within a sample. Cell activation is required for the production and release of most cytokines.
Yes, up to 3 surface markers can be detected. IsoPlexis provides validated surface marker staining cocktails for use with the IsoLight system. Read more here.
The answer is sample-dependent: For frozen PBMCs and CAR-T pe-infusion product we typically recommend 1-2x106 total cells which roughly translates to 1-2x105 T cells. For cells from limited sources, e.g. TILs, we have gone as low as 10,000 cells. Our standard protocol recommends loading 35ul of 1.25x106 cells/ml suspension which translates to ~45,000 cells per chip.
I need to use less than the recommended amount of cells. Should I change the loading volume or cell concentration?
The IsoCode chip requires 30-35 ul of cell suspension. Simply resuspend your cells in 30 ul of complete media and load onto the chip as instructed.
I would like to flow sort my cells using an antibody that is not part of your cell marker staining cocktails. How do I go about doing this?
Please refer to our surface staining guide (coming soon) for details on compatible fluorophores that can be used in conjunction with our validated surface staining cocktails.
I am using magnetic sorting to enrich for my cell population. Do I still need to perform surface staining?
Yes, in order for the assay to perform properly, cells have to be stained with one of IsoPlexis' staining cocktails and according to the supplied cocktail-specific protocol.
Yes, customers routinely run our assay on previously frozen PBMCs or CAR-T cell product. Suggested protocols for cryopreserving and thawing of PBMCs will be available soon.
What concentration of LPS should be used for stimulating monocyte assays, and how long should the cells be stimulated for?
Are you able to work with unfixed blood and cells from bacterially infected animals/tissues or infectious agents themselves (bacteria, viruses)?