Frequently asked questions (FAQ)
The volume of blood per test depends on your application and expectations. Here are some extreme examples:
Example#1: You plan a deep analysis by mass cytometry, expect to find small populations with ≈1:10.000 frequency and you need at least 500-1000 cells/population to make the statistics valid enough:
• 500 cells/population x 10.000 = 5.000.000 cells
• efficiency at acquisition of CyTOF ≈30-50% => ≈15.000.000 cells
• doublets rate, cell losses in processing => 25-30.000.000 cells.
At standard blood count ≈5-7.000.000 leukocytes/ml of blood => you will need at least 4-5ml of blood.
Example#2: Flow cytometric analysis of standard cell populations where e.g. some DCs, NK cells being the smallest ones with ration ≈1:100-200; you plan only one panel (tube) and you need 500 cells per population:
• 500 cells/population x 200 = 100.000 cells
• efficiency at acquisition ≈97-100%
• doublets rate or cell losses in processing, extra volume in acquisition => 200-300.000 cells.
At standard blood count ≈5-7.000.000 leukocytes/ml of blood => you will need 50-100 µl of blood.
Details to consider:
1. Targeting very rare populations with mass cytometry is technically very demanding and expensive as it takes a lot of machine-time, antibodies etc. Targeting them with flow cytometry is faster and cheaper but requires more knowledge on what you are looking for.
2. Storing more blood (or more aliquots of the same sample) in Stabiliser and/or aliquoting after fixation allows you to bio-bank an aliquot or do more analysis later (e.g. in situation when journal reviewers ask for more analyses).
Storing 500µL - 1mL of blood is a cost-effective solution that allows you to save at least one aliquot as a "fail-save" and do the most of standard applications. Cell counts recovery rate in processing can be virtually 100% with proper laboratory practice. Some losses are inevitable in last steps of sample washing with diH2O prior to mass cytometry acquisition, though.
Cytodelics Whole blood processing kit is compatible with the barcoding protocol but you should skip the fixation step and start with the permeabilisation step.
Since the "Whole blood processing kit" is primary designed for "thick" samples, you can concentrate samples by gentle centrifugation before fixation in cases when RBCs count is relatively low. If you are taking cell counts before fixation, your target cell density should be ≈10-40x106/mL. But if the sample's RBCs count is high you should proceed without concentrating the sample. All other steps are given by the sample volume.
For cell cultures samples or PBMCs, please, try our kit specifically designed for this type of samples.
