Cell Culture Techniques 程洪强，方瑜，孙明姣 2013年12月

Cell passage, freezing and proliferation assay

Procedures for Cell Passage Warm media and trypsin in 37C water bath. Check cells in T flask under microscope to confirm that the cells are 90%-100% confluent. Clean and sterilize. Remove cell plates from the incubator and quickly place in hood. Empty liquid media covering cells Add 10 mL of PBS to the plate, gentle rinse the cells and remove. Add 1 mL trypsin to the plate. Lightly swish trypsin. Place flask in incubator for 5 min, or until detached. Visually check to ensure lumps of cells are dispersed. Check cells under microscope to confirm that cells are detached from the surface. Add 6 mL of media to dilute trypsin. Carefully resuspend cells. Put in 15 mL centrifuge tube. Centrifuge After centrifugation, aspirate supernatant. Cell pellet should remain at base of tube. Resuspend cells in 8 mL of media. Aliquot appropriate volume of cell suspension into freshly prepared plates with media Mix and place the plates in incubator.

Procedures for Freezing Trypsinize cells (standard protocol). Re-suspend cells in media, transfer to a sterile centrifuge tube, centrifuge at 1000 RPM and 4C for 3-5 min. Remove supernatant with sterile pipette. Quickly re-suspend pellet by adding 1 ml freezing media per vial to be frozen. Aliquot 1 ml freezing media plus cells per vial, and place on ice. Freeze overnight at -80C. Transfer vials to liquid N2 tank for indefinite storage.

Procedures for Cell Proliferation trypan blue staining MTT 3H-TdR CFSE 5. iCELLigence /xCELLigence system

Procedures for iCELLigence 1. Click the icons to select a experiment process: New experiment Protocol Cell QC Standard Protocol 2. Add 150 ul medium in each well of E-plate L8.

3. Place E-plate L8 on iCELLigence , the system will automatically scan, if the contact is good, go to next step. 4. Set the cell name and number on the layout page. 5. Click the Start button at the left bottom to detect the baseline. 6. Take E-plate out and add 300 ul prapared cell suspension in each well. 7. Left the E-plate at room temperature for 30 min. 8. Place the E-plate on the iCEELigence equipment In the incubator.