Question: How should I count my cells?
Answer: We recommend the following cell counting methods:
- Countess II Automated Cell Counter (Thermo Fisher), cell size range 7-60 um, limited in measuring the viability of very small cells (discontinued)
- This has been replaced by the Countess 3 Automated Cell Counter (Thermo Fisher). The updated counting algorithm for Countess 3FL is more aggressive at gating out debris, defined as particles <4uM. This can be problematic for isolated nuclei or certain cell types, such as small 5-7µm T cells, which may be excluded from the final count, leading to inaccurate cell loading concentrations.
- Hemocytometer, any cell size range
- Cellaca MX High-throughput Automated Cell Counter (Nexcelom), cell size range 4um -25um, up to 24 samples at time, pre-programmed settings for counting specific cell types are available (ie. for PBMCs, we use Cellaca Cell Setting MX04.1_Human_PBMC_F1)
Accurate cell counting is critical to application success.
Please refer to the appropriate Single Cell Gene Expression or Immune Profiling User Guide for appropriate cell stock concentration and loading volume to maximize the likelihood of achieving the desired cell recovery target. If possible, bring the input cell/nuclei suspension to a concentration that is optimal for the dynamic range of the counting technique used (manual or automated), and allows for 3 – 4 reproducible counts (where the standard deviation of these counts is <25%).
We recommend counting after sorting, unless cell recovery is very limited.
|
Dye |
Mechanism of Action |
Useful for |
Fluorescence Needed |
|
Trypan Blue |
Cell impermeable, will stain dead cells or nuclei |
Cell or nuclei counting |
None |
|
AO/PI |
AO: Cell permeable, stains all nucleated cells green PI: Cell impermeable, will only stain dead cells or nuclei red |
Good for distinguishing between cells (live vs dead) and debris. |
Yes |
|
Calcein AM |
Cell permeable, will stain all nucleated cells |
Total cell count, distinguishing cells from debris. |
Yes |
|
Ethidium homodimer-1 |
Cell impermeable, will only stain dead cells or nuclei |
Counting nuclei only, measuring cell viability |
Yes |
For additional information on obtaining accurate cell counts, see the related Technical Note (https://support.10xgenomics.com/single-cell-gene-expression/index/doc/technical-note-guidelines-for-accurate-target-cell-counts-using-10x-genomics-single-cell-solutions)
Products: Single Cell Gene Expression, Single Cell Immune Profiling