How to subculture (passage) primary cells

Gather all materials in the biosafety hood and set up instruments. Prepare basal media and supplements according to product instructions, then label and date all medium bottles.

Examine the cells under the microscope to verify that confluency is approximately 80% and that mitotic cells are present before proceeding with subculture.

Remove all culture media from the flask, add 9 mL of trypsin EDTA solution and rock the flask to cover the entire surface. Then remove all 9 mL and add 3 mL of fresh trypsin EDTA solution.

Incubate the flask at room temperature until cells become completely round (1-20 minutes depending on cell type). Gently rap the flask to dislodge any remaining adherent cells, and use microscopy to confirm complete dissociation.

Add 9 mL of trypsin neutralizer to the flask and transfer the cell suspension to a 50 mL conical tube. Pipette another 9 mL of neutralizer over the flask surface to remove remaining cells and add to the tube.

Centrifuge cells at 180 G for 7 minutes, then carefully remove the supernatant without disturbing the pellet. Resuspend the cell pellet in 12 mL of supplemented medium and gently pipette to mix well.

Determine cell concentration in the suspension, dilute cells in supplemented medium to the appropriate density, and seed new culture vessels. Return the vessels to the incubator for continued culture.