Product name: 293T-CD3mix-puro cell

Preservation system: long-term storage in liquid nitrogen

Culture conditions: 85%DMEM+10%FBS+5%DMSO

Growth type: adherent growth

Transport method: frozen cells transported on dry ice

Recovery process:

1. Put on a protective mask and antifreeze gloves, remove the cells from the liquid nitrogen tank, and quickly put them in a 37°C water bath to thaw.

2. After the cells are thawed, the cells are delivered to the cell chamber through the delivery chamber.

3. In the biological safety cabinet, take out a 15mL sterile centrifuge tube, and use a 1mL pipette to add 4mL medium to the 15mL centrifuge tube.

4. Use a 75% medical disinfectant alcohol cotton ball to carefully wipe the outer surface of the cell cryopreservation tube that has been thawed in a 37°C water bath for surface disinfection. In the biological safety cabinet, unscrew the cell cryopreservation tube, then use a 1mL pipette to aspirate the cell suspension in the tube, and add it dropwise to the 15mL centrifuge tube containing 4mL medium prepared above, and screw the lid of the 15mL centrifuge tube. Tightly, slowly turn upside down 4 times to mix the cell suspension.

5. Place the centrifuge tube containing the cell suspension in the centrifuge and set the centrifugation parameters as follows: Centrifuge at 200xg at room temperature (~25°C) for 5 minutes.

6. After centrifugation, gently transfer the centrifuge tube to the biological safety cabinet. At this time, white cell clusters can be seen at the bottom of the cytocentrifuge tube. Use an electric aspirator to aspirate the medium supernatant (the cell cluster is relatively loose, be careful not to touch the cell cluster at the bottom).

7. Use a 1mL pipette to add 2mL of complete medium, gently pipette 5 times to resuspend the cell pellet, count, and inoculate.

8. Place the cells in an incubator for overnight culture.

  Illustration: Left: use APC-anti-CD3 antibody (Biolegend, Cat#317318) to detect CD3 expression;

Right: TCR expression plasmid was transiently transfected, and PE fluorescently labeled tetramer HLA-A 02:01 HPV16 E7 Tetramer (MBL Cat#TB-0031-1) was used to detect the expression of TCR.