I tried to culture the DU145 cell twice from the beginning (stock from the Nitrogen tank).
The first time I tried EMEM + FBS and the second time I tried RPMI.
both efforts ended up not growing.
I tried a mycoplasma test, small flask, and centrifuge at each time splitting, but still they do not grow!
if you have any idea about any of the steps or anything, Please share it with me.
Thank you in advance.
Hello Sahel Shemshadi
You may incorporate the following steps in the thawing protocol. In case, you have missed certain steps!
1. You should follow a quick thaw process. Hold the cryovial on the surface of the water bath at 37 deg C and occasionally flick it gently during thawing. Do not leave the cryovial unattended during the thawing process because thawing takes only a minute or two.
2. You may add warm complete EMEM medium drop-by-drop into the cryovial containing the cell suspension, slowly over a 40-second period. The final volume should be around twice the volume of the cell suspension (for instance, add about 1 ml of complete EMEM medium to a cryovial containing 1 ml of cell suspension). Be careful not to exceed the capacity of the cryovial.
3. Transfer the diluted cell suspension into a 15ml centrifuge tube containing 8ml of warm complete EMEM medium. For cells preserved in a freezing medium containing DMSO, it is recommended to wash out or dilute the DMSO immediately post-thaw because DMSO harms the cells at higher concentration.
4. Centrifuge the cell suspension at low speed 250g for 7 minutes to prevent cell damage.
5. Decant the supernatant and tap the cell pellet.
6. Resuspend the cells in a desired volume of warm complete EMEM medium and take the cell count, which will give you an idea of percent viability after thawing. Resuspend the cell suspension with the recommended volume of complete medium into a T -25 cm^2 culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. Therefore, it is suggested that prior to the addition of the vial contents, the culture vessel containing the EMEM complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.4).
Please note: If you feel that the cells are not sturdy and may get damaged during the centrifugation step, you may transfer the entire content of the vial in T-25 cm^2 culture flask along with complete medium (up to 5-10ml). The next day or when the cells have attached to the substratum, you may change the medium with fresh complete medium to get rid of DMSO.
Hope this helps!
Good Luck!
Malcolm Nobre
Thank you for your reply!
it was so complete and helpful. I will try again and employ your tips.
Good luck
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