Protocol For Thawing Cryopreserved Leukopaks (RUO) For Highest Viability
Recommended method for handling and thawing
September 30, 2021
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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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