Thawing (XB10) hybridoma cells

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  • 1 x 15 ml conical tube per vial of frozen cells
  • water bath (37 C for mammalian cells)
  • High serum media (RPMI media +10% FCS+glutamine+P/S) [Use Defined Supplemented Calf Serum (DSCS) from Hyclone (Prebio) for making high serum media for hybridoma cells]
  • 1 blue cap flask
  • 1 x 10 ml pipettes
  • gestopfft pasteur pipettes
  • ungestopfft pasteur pipettes
  • frozen cells in liquid nitrogen or dry ice


  1. Put 9 mls of HS RPMI into the conical tube
  2. Thaw the cells in the water bath until there is a very small piece of ice left.
  3. Immediately after, use the gestopfft paseur pipette to transfer the cells into the conical tube. Do not allow cells to sit in the freezing media very long. If thawing more than 1 vial, thaw one vial at a time and put the cell into the 9 mls of media right away.
  4. Spin at 1000 rpm for 3 minutes
  5. While waiting put 10 mls of media into the blue cap flask.
  6. When the cells finish spinning, aspirate off the media in the tube
  7. Bang the tube on the workbench to resuspend the cells
  8. Add 2 mls of HS RPMI to the conical tube and resuspend the cells
  9. Transfer the cells from the conical tube into the flask and rock the flask to distribute the cells
  10. Put cells in 37 C and 5% CO2. Label the flask with the name of cells, the passage number, your name and the date
  11. On the next day check the cells. These cells remain round, and many stay floating. They should have smooth edges and are bright under the microscope. It is not good if they have rough edges and are “phase dark” under the microscope.