Re: 2014-Yao et al-J Immunol Methods
Hi VIncius,
Length of acquisition depends slightly on the instrument version:
1. CyTOFv1 and CyTOFv2 with standard sample loops have a flow rate of 45uL/min and a cell transmission efficiency of ~20%.
2. CyTOFv1 and CyTOFv2 with a SuperSampler have a flow rate of 45uL/min and a cell transmission efficiency of ~40%. So, in theory, it would take half the amount of time to acquire the same number of cells. In practice, you often need to dilute your sample slightly more because of background, etc, so your time savings might not be half but maybe only 2/3 of the regular sample loop time.
3. At least currently, Helios (CyTOFv3) have a flow rate of 30uL/min and a transmission efficiency of ~40%. You get similar numbers if you use a SuperSampler on a Helios. Again, because of additional dilution (background, clogging, etc), you actually don't save much time relative to the v1 or v2 at the faster flow rate.
In short: to acquire 200-300K Ungated (found) cells, you're typically looking at ~20min acquisition time. Of that, you'll wind up with ~150K Live Intact Singlets (ie, all the garbage gated out and you're ready to do all your marker-based gating).
Regarding antigen-specific T cells: as I said in a previous post in this chain, their frequency is often reported as per 1 million CD4+. Therefore, you might need to collect several million cells in order to get your tens to hundreds of antigen-specific cells. Of course, this depends on the frequency in the sample: a normal healthy person might have a very low level of antigen-specific cells, whereas someone with an active infection or an active autoimmune condition might be much higher. You can often find approximate numbers in the literature to help you calculate your target number of cells, and whether enrichment might be necessary.
Mike
Length of acquisition depends slightly on the instrument version:
1. CyTOFv1 and CyTOFv2 with standard sample loops have a flow rate of 45uL/min and a cell transmission efficiency of ~20%.
2. CyTOFv1 and CyTOFv2 with a SuperSampler have a flow rate of 45uL/min and a cell transmission efficiency of ~40%. So, in theory, it would take half the amount of time to acquire the same number of cells. In practice, you often need to dilute your sample slightly more because of background, etc, so your time savings might not be half but maybe only 2/3 of the regular sample loop time.
3. At least currently, Helios (CyTOFv3) have a flow rate of 30uL/min and a transmission efficiency of ~40%. You get similar numbers if you use a SuperSampler on a Helios. Again, because of additional dilution (background, clogging, etc), you actually don't save much time relative to the v1 or v2 at the faster flow rate.
In short: to acquire 200-300K Ungated (found) cells, you're typically looking at ~20min acquisition time. Of that, you'll wind up with ~150K Live Intact Singlets (ie, all the garbage gated out and you're ready to do all your marker-based gating).
Regarding antigen-specific T cells: as I said in a previous post in this chain, their frequency is often reported as per 1 million CD4+. Therefore, you might need to collect several million cells in order to get your tens to hundreds of antigen-specific cells. Of course, this depends on the frequency in the sample: a normal healthy person might have a very low level of antigen-specific cells, whereas someone with an active infection or an active autoimmune condition might be much higher. You can often find approximate numbers in the literature to help you calculate your target number of cells, and whether enrichment might be necessary.
Mike