Sequential isolation
The Donor Difference
Counting protocolCryopreserved leukopak thawing
A consistent thawing method that protects quality, viability and purity, and makes results repeatable. Count cells immediately after thawing, before any other manipulation.
What you need
Reagents
- Thawing medium: RPMI 1640, IMDM or DMEM + 10% heat-inactivated FBS, at room temperature (20–25 °C)
- Optional: DNase I 0.1 mg/mL to reduce clumping
- 70% ethanol
- Resuspension medium for your application
Equipment
- 37 °C water bath
- Biosafety cabinet
- Sterile 100–250 mL conical tubes
- Centrifuge capable of 300 g at 4 °C, brake off
- Cell counting and viability setup
Thawing procedure
Prepare
Place thawing medium in a 37 °C water bath, disinfect the container with 70% ethanol and move it into the biosafety cabinet.
Thaw
Take the cryobag from storage and place it immediately in the 37 °C bath, fully submerged and still. Invert gently to check progress, then return it.
Disinfect and mix
As soon as it is fully thawed, remove the bag, disinfect with 70% ethanol, move into the cabinet and invert gently twice.
Transfer
Empty the bag through its port into a sterile 100–250 mL conical. Rinse the bag with 5–10 mL thawing medium and add the rinse.
Dilute
Slowly add thawing medium up to 4× the cell suspension volume. Cap, invert gently 3–5 times, and take a sample for count and viability.
Spin
Centrifuge at 300 g for 20 minutes at 4 °C, no brake. Confirm a visible pellet.
Aspirate
Slowly aspirate and discard the supernatant without disturbing the pellet.
Resuspend
Flick to loosen the pellet, then slowly add medium to the desired volume and concentration. Confirm count and viability.
Safety precautions
Health hazards of liquid nitrogen
LN2 expands about 700-fold, which can burst cryovials, containers or equipment. Contact with LN2 or cold vapor causes severe tissue damage. Nitrogen displaces oxygen and can cause dizziness, nausea, unconsciousness and death.
- Wear waterproof, thermal-insulated cryo gloves (never submerge hands), lab coat, long pants, closed-toe shoes and chemical splash goggles
- Supplied cryovials are for vapor-phase storage only
- For liquid-phase storage use approved liquid-phase cryovials, or seal gas-phase vials in an LN2-rated outer envelope
- Handle cryovials in a biosafety cabinet or chemical hood with the sash lowered