Hi Joanna,
I think there are two things in your one question:
1. Viability (and to a lesser extent, singlets).
2. Cell count.
1. Viability: This is also in 2 parts: how long it takes you to acquire however many LiveIntactSinglets (poorer quality sample = longer acquisition), and the impact of poor sample quality on proper staining (somewhere around 60% viability, the staining gets *noticeably* worse even in the LiveIntactSinglets).
2. Cell count: I think it comes down to what your question is, and the rarity of your cell population. In another thread (
viewtopic.php?f=6&t=1980&p=5160) , Cynthia Guidos talked about and gave a calculator for counting statistics ("You need to decide on how precise you want your measurement to be and to estimate the frequency of the rarest subset that you want to measure").
So, if you're looking at a phospho panel (eg, pSTAT1), in most cases an entire parent population such as CD4+ will respond to the stim. Put differently, the entire CD4+ pSTAT1 histogram will shift to a higher signal. Since the entire population is responding, you have more events to satisfy your Cell Count.
In contrast, for ICS, maybe only *some* of your CD4+ will make a particular cytokine upon stim. So, you'd need proportionately more CD4+ (and therefore total Cell Count) in order to satisfy Cell Count for IFNg+ CD4+.
In an extreme case like antigen-specific T cells (peptide stim, or tetramer), you may need to collect a *lot*.
In a good viability PBMC sample, a rule of thumb at the Stanford HIMC we aim for:
1. surface pheno (no ICS): 250K Ungated/sample -> ~150K LiveIntactSinglet.
2. ICS (surface + cytokine): ~300K Ungated/sample -> ~200K LiveIntactSinglet.
3. Phospho (surface + phospho transcription factors): ~150K Ungated/sample -> ~100K LiveIntactSinglet.
Mike