Posted 14 November 2011 - 10:17 AM
I am using HK-2 (human kidney proximal tubule immortalized cells). I am using low glucose DMEM with 10% FBS and 1% Penn-strep for their maintenance as reported in several papers. I am finding it extremely hard to detach these cells from the bottom of the flask. I have tried washing with 1 ml trypsin prior to PBS, incubation at 37C for 10 minutes, pipetting vigorously with trypsin but nothing really works. There remains almost entire cell bed at the bottom of the flask.
Does anyone have an experience of handling these cells or any other suggestions?
Posted 05 January 2012 - 12:33 PM
So: Are you using trypsin/EDTA? Are you rinsing the cells before adding trysin/EDTA? Have you tried leaving them longer? Who has cultivated these cells before you (and did they experience the same problems)? Have you been splitting these cells a lot or over a long period?
Growth Conditions: Cell growth is dependent on epidermal growth factor. The cells should not be allowed to become confluent. Subculture at 80% of confluence.
Note: The cells should not be allowed to become confluent, subculture at 80% of confluence.
Remove and discard culture medium.
Briefly rinse the cell layer with 0.05% (w/v) Trypsin- 0.53 mM EDTA solution.Add 2.0 to 3.0 ml of 0.05% Trypsin-0.53 mM EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 xg for 5 to 10 minutes.Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:4 is recommended
Medium Renewal: Every 2 to 3 days.