Cell culture
Flow cytometry staining
Work out the preparation quantities for a staining run: total cells and buffer for a set of tubes, antibody per test from its titre, the cocktail total, and how long acquisition will take. These are amounts, not a protocol; the panel and titres come from your own validation.
Antibody per test
From a dilution, antibody per test is the stain volume divided by the fold. From a mass per test, it is that mass divided by the stock concentration. The total carries the tube count and overage so a cocktail covers every tube. Titres are specific to each antibody lot — determine them by titration rather than assuming.
Acquisition time
These are preparation quantities, not staining protocols. Cells per tube, stain volume and titre come from your panel and your validation; the tool scales them to the number of tubes and adds the overage you set. Acquisition time is simply the target events divided by the event rate.
Formula
antibody per test = stain volume ÷ dilution (or mass per test ÷ stock concentration)
- cells per tube
- the number you stain and acquire per tube
- stain volume
- the volume the antibody is added into per tube
- titre
- given as a dilution, 1:X, or as a mass per test against a stock concentration
- overage
- a margin so a shared cocktail covers every tube
Worked example
A 1:200 antibody across 12 tubes in 100 µl
- 1 × 10⁶ cells per tube
- 12 tubes, 10 per-cent overage
- 100 µl stain, titre 1:200
- Antibody per test: 100 over 200 is 0.5 µl.
- Working factor: 12 times 1.10 is 13.2 tests.
- Antibody total: 0.5 times 13.2 is 6.6 µl.
Answer 0.5 µl per test, 6.6 µl of antibody for the cocktail
Common mistakes
- Using the vendor’s suggested dilution as a titre. The optimal amount is per lot and per instrument; titrate to the best signal-to-background rather than trusting the label.
- Sizing a cocktail with no overage. Cocktails cost the most reagent and lose volume to every tube and tip; a margin keeps the last tube from missing an antibody.
- Staining at the wrong cell number. Too many cells per fixed antibody amount drops the effective titre; keep cells per tube consistent with how you titrated.
Frequently asked
Dilution or mass per test?
Whichever your titration produced. A dilution is quick when the stock is fixed; a mass per test is clearer when stocks vary between lots.
Is acquisition time exact?
It is target events divided by event rate, so it is only as good as your estimated rate. Rare populations and clogs make the real time longer.
Does this design compensation?
No. It handles the quantities of a stain; controls and compensation are a separate part of setting up the experiment.