Núria Crua· Postdoc· University of Lausanne (UNIL)
Published Jul 27, 2026~35m total· 1 contributions
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JNAR-ID: JNAR-03477264
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PublishedLast updated Jul 27, 2026
Objective
Protocol for thawing the THP-1 cell line, including washing out the DMSO used for cryopreservation and re-establishing the cells in culture.
Classification
Sample typeCells
TechniqueCell Culture
Experimental contextIn vitro
Biological modelCell LinesHuman
Reproducibility status
Reproducible
No reproducibility blockers or risks reported. The protocol runs end-to-end.
8 healthy
⏱ Total 35 min1 section8 steps
Thawing of the THP-1 Cell Line
35 min
1
Prepare the gowing culture medium: RPMI 1640 + 10% heat-inactivated fetal bovine serum + 1% antibiotic (Penicillin/Streptomycin/Amphotericin or Gentamicin).
2
Before retrieving the vial from liquid nitrogen, prepare 3 Falcon tubes with 20 ml of culture medium and warm them in a 37 °C incubator for f 15 minutes15 min
Critical stepWarm the medium for no more than 15 minutes to prevent it from being adversely affected.
3
Using a 1000 µl pipette, take 0.5 ml of prepared medium from one of the falcon tubes and slowly thaw the cells from the vial.
TipThaw slowly, drop by drop, and do not touch the frozen cells, as they could easily die.
4
Centrifuge for 10 minutes at 1500 rpm to wash out the DMSO used for freezing10 min
5
Extract and discard the supernatant by decanting
Critical step
WarningDecant only once; decanting more than once would result in loss of the cells
6
Resuspend the remaining cell pellet by gently tapping with a finger and add 20 ml of the prepared medium from the second falcon tube.
7
Centrifuge again for 10 minutes at 1500 rpm for a second wash and discard the supernatant.10 min
8
Resuspend the cell pellet by gently tapping and add the correct volume of cell medium
TipIf this is the first passage and the cells have recently arrived at the lab from the commercial supplier, or if the cell concentration is low after counting, add 5 ml of medium and place them in a small flask (25 cm2) in the 5% CO2 incubator at 37 °C.
TipIf it is a vial already frozen from a more advanced passage with a high cell concentration, it can be resuspended in 20 ml and a 75 cm2 flask used under the same incubator conditions.
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