Cytomaton @Cytomaton

CS&T vs Rainbow vs EQC comparison, specific thresholds, and troubleshooting bead failures: cytomaton.ai/blog/flow-cyto…

Daily startup: FACS Clean 5 min, DI water rinse, 20-30 min laser warm-up, 10,000 bead events, review before samples. Most skipped step: laser warm-up. Unstabilized lasers inflate %rCV by 0.5-1%.

Three bead types for daily flow QC. CS&T: automated, BD-specific, PMT voltage auto-set, flags at +/-50V. Rainbow: manual, instrument-agnostic, ideal for cross-instrument harmonization. EQC: for labs in proficiency programs requiring external benchmarks.

Doublets sneak through scatter gates and inflate CD8+ counts by 3-8 points. The fix is a 20-second FSC-A vs FSC-H gate placed before viability, not after. cytomaton.ai/blog/doublet-d… #flowcytometry

Three bead types, three different jobs in flow cytometry QC. CS&T auto-adjusts and tracks per BD protocol. Rainbow beads work on any instrument. EQC beads for proficiency programs. When to use each: cytomaton.ai/blog/flow-cyto… #flowcytometry

Full titration protocol, SI calculation, how to read the curve, and when to re-titrate after new lots or cell type changes: cytomaton.ai/blog/antibody-…

Critical: titrate on the actual cell type at the actual cell density you will use. Moving from fresh to frozen PBMCs can shift optimal concentration by a full dilution step. The vendor optimized on their reference cells, not yours.

Vendor antibody concentrations for flow cytometry: you are probably using 30-50% more than you need. Not their fault. Their reference cells, their instrument. Not yours.

Your vendor antibody recommendation for flow cytometry is typically 30-50% higher than optimal. The Stain Index shows exactly where the plateau is. Titration protocol: cytomaton.ai/blog/antibody-… #flowcytometry #paneldesign

How to set up the chart, which Westgard rules apply for flow, and what gradual %rCV creep tells you before a detector fails: cytomaton.ai/blog/flow-cyto…

The Levy-Jennings fix: baseline 20 sessions, compute mean and SD per channel, then chart MFI and %rCV each run. Westgard rule that matters most: 10 values in a row on the same side of the mean. Nothing flags. Instrument is drifting.

Your CS&T bead run passed. Tuesday and Thursday: green. Then Friday a batch of 40 samples came back with the CD4/CD8 ratio shifted 18%. The report was still green. Here is what was actually happening.

All four compared on fixation compatibility, brightness, channel cost, and panel impact: cytomaton.ai/blog/live-dead…

Decision rules: fixing samples? Fixable, always. UV laser available + want PE/PerCP slots free? DAPI. Live cells, simple panel, no PE conjugates? PI is the cheapest right answer. Spectral panels over 15 colors? Fixable amine dyes.

Choosing a live/dead stain looks simple — until you realize PI takes your PE channel, DAPI needs a UV laser you may not have, and fixable amine dyes are 5-10x the cost but survive paraformaldehyde fixation.

Your CS&T bead run passes every day. Meanwhile your PE-channel PMT voltage has drifted 32V in two weeks. Here is how to track flow cytometry QC metrics across runs with Levy-Jennings charts. cytomaton.ai/blog/flow-cyto… #flowcytometry