Protocol For Thawing Cryopreserved Leukopaks (RUO) For Highest Viability
Recommended method for handling and thawing
September 30, 2021
FOR RESEARCH USE ONLY. NOT INTENDED FOR USE IN DIAGNOSTIC PROCEDURES.
Detailed Instructions for Thawing Discovery Cryo Leukopaks™ (RUO)
MATERIALS NEEDED FOR DISCOVERY CRYO LEUKOPAKS (RUO) THAWING:
- HBSS (w/o calcium or magnesium) + 10% FBS + 0.1 mg/ml DNase (Thawing Buffer)
- ** We recommend the use of DNase to prevent cell clumping due to DNA leakage from dead cells.**
- 70% Ethanol
- Discovery Cryo Leukopak (RUO)
- Sterile bottle
- Sterile scissors
- Disposable pipettes and micropipette tips
EQUIPMENT NEEDED FOR DISCOVERY CRYO LEUKOPAKS (RUO) THAWING:
- Biological Safety Cabinet
- Water Bath at 37°C
- Serological and Micropipettes
- Liquid Nitrogen Freezer
- Automated Cell Counter
- Warm HBSS + 10% FBS in 37C water bath. Wipe with 70% ethanol and transfer into the
biological safety cabinet.
- Remove Discovery Cryo Leukopak (RUO) from liquid nitrogen storage. Immediately place in a 37°C water bath and fully submerge. Do not move the leukopak while it is thawing.
- Once only a small ice crystal remains, remove the leukopak from the water bath, thoroughly clean the outside of the bag with 70% ethanol, and transfer into the biological safety cabinet.
- Using sterile scissors, cut the port on the leukopak and slowly transfer the cell suspension into an appropriate sterile bottle.
- Add an equal volume of Thawing Buffer to cell suspension dropwise.
- Add one-half volume of Thawing Buffer to the original leukopak, thoroughly mix, and transfer to cell suspension.
- Gently add an additional two volumes of Thawing Buffer to the cell suspension.
- Thoroughly mix the sample, and take a small sample for cell count and viability.
- Samples are ready to be centrifuged and downstream analyses.
- Add DNase to 0.1 mg/ml final concentration.
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