Sequential isolation
The Donor DifferenceCounting cells and percent viability
Count fresh products immediately on receipt, and cryopreserved products immediately after thawing, before any other manipulation. Discovery is not liable for cell loss during later processing.
What you need
Recommended: automated counter
- Dual nucleation dye such as acridine orange / propidium iodide
- Cell counting chamber
- Automated cell counter
Alternative: trypan blue
- 0.4% trypan blue
- Neubauer hemacytometer and coverslip
- DPBS (optional, for dilution)
Recommended method
Mix thoroughly
Mix the suspension so cells are evenly distributed.
Fresh product in a bag
Hold the bag by its top and bottom edges and invert side to side, about 1 second per rotation, 5 rotations.
- Wipe the transfer line with an alcohol swab, clamp it, cut with sterile scissors
- Unclamp and dispense a 2–3 mL aliquot into a sterile tube
- Pipette up and down at least 5 times with a P1000
- Dilute for your counter, e.g. 100 µL sample + 900 µL DPBS (1:10) for Cellometers
Fresh product in other containers
Gently pipette 25–50% of the suspension up and down at least 5 times, then dilute as above.
Cryopreserved product
Thaw first using the cryo leukopak or vial procedure.
Stain
Mix the cell suspension with an equal volume of counting dye.
Load and count
Load the chamber for your counter and count per the manufacturer's instructions, accounting for the initial dilution.
Alternative method with calculator
Aliquot
Mix and immediately take a 10 µL aliquot.
Stain
Combine 10 µL sample with 10 µL 0.4% trypan blue. Count within 5 minutes.
Load
Load about 10 µL into the hemacytometer, filling the chamber.
Read
Under bright field, dead cells stain light to dark blue; viable cells are clear.
Count
Count viable cells in one quadrant; aim for ≥100 viable cells, using more quadrants if needed. Count dead cells in the same quadrants.
Count at least 100 viable cells for an accurate result.
Concentration = (viable ÷ quadrants) × dilution × 10⁴. Viability = viable ÷ (viable + dead) × 100. Dilution 20 = 1:10 pre-dilution × 1:1 trypan blue.