Tissue Culture Series #1: How to Thaw Cells with High Efficiency

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Tutorial demonstrating best practices for starting a fresh cell culture from frozen stocks, including cell thawing tips and tricks to maximize survival.
• Ice crystals and thaw time
• How to avoid osmotic shock
• Cryoprotectant removal and media replacement
• Thawing stem cells and primary cells
• Methods for resuspending cells
• Seeding density considerations
• Troubleshooting

[@funny11744]

Regarding using the antibiotics for primary cells : Does is it necessary to add antibiotics whenever we change the media( let us say every 24 hours) for the first passage P0 , or it is recommended to add antibiotics just for the first 24 hours ?

[@misscarebearclaire418]

I was told that if you didnt do a second spin down/resuspension that there would be remaining DMSO left over and cause cell damage. Is this true? or is that extra spin causing unneccessary stress

[@user-tp6vx5kk8m]

I could not understand what exactly happens after aspirating cells. Please explain.

[@milliporesigma2407]

After aspirating the wash media, gently resuspend the remaining cell pellet in a small volume of pre-warmed culture media by pipetting up and down. Suspension volume and flask size is determined by the number of cells that were recovered.