Question: Are the cell counting recommendations different for the Fixed RNA Profiling assay?
Answer: Accurate sample counting is critical for optimal assay performance for the Fixed RNA Profiling assay. When preparing a single cell or nuclei suspension, customers should continue to use our best practices for optimal cell counting.
It is strongly recommended that the sample be stained with a fluorescent dye such as ethidium homodimer-1 or AO/PI staining solution and counted using an automated fluorescent cell counter (Countess II Automated Cell Counter or a Cellaca counter) for accurate sample counting. The use of fluorescent dye during cell counting enables accurate quantification even in the presence of sub-cellular debris. The optimal cell concentration for the automated cell counter is 1,000-4,000 cells/μl. Please refer to manufacturer's instructions for details on operations. Additional details and guidelines provided in Demonstrated Protocols and User Guides on the 10x Genomics Support site.
Please note: Countess III is currently incompatible with small cell/nuclei counting due to software limitations which gates out cells/nuclei < 5 microns.
Debris-free samples (cells or nuclei suspensions) can also be counted using trypan blue. Trypan blue is strongly not recommended for samples with any sub-cellular debris present.
Post-Hybridization Cell Counting: Accurate cell counting is critical for optimal assay performance. When using fluorescent dyes, signal to noise ratio may be lower because of the Conc. Post-Hyb Buffer composition. Increasing the dilution or a serial dilution into PBS before counting may improve the signal to noise ratio.
Products: Fixed RNA Profiling Gene Expression