Hello,
I am new to cancer research and I am trying to optimize my small cell lung cancer cell protocol.
I purchased a cell line from ATCC (H1105) and I have been growing the cells in RPMI 1640 media (10% FBS + penicillin/streptomycin.
I am looking for anyone who was worked with these cells in the past and who can let me know if these cells look normal?
I noticed the cells grow very slowly and form spheres.
Am I using the right media? How can I get the cells to grow faster? Can someone refer me to a good SCLC cell culture protocol? Thank you.
Till now your cells looks fine. If you want to grow the cells faster you can increase the concentration of the FBS.
You can also try DMEM medium.
Good luck!
These cells are a clustering floating cell-line, so clumps are part of the phenotype. Confluence-wise they are not that confluent yet as these form large clusters when confluent, how many days were they in culture? The medium you are using is not the recommended one which is a HITES medium supplemented with 5% FBS. Check ATCC (https://www.atcc.org/products/all/CRL-5856.aspx#generalinformation). If in doubt, do a trypan blue viability stain and determine the ratio of dead cells, it could be that the medium is incompatible with growth.
Thank you Mohd and Rabeah,
I have had these cells for several weeks. I am adding media every 3-4 days and passaging every 10. Still the cells are growing pretty slowly.
How would you recommend harvesting the cells for passaging? I feel I am losing some during this time. Do you recommend Typsin or Accutase? At what speed do you spin the cells? These small details I cannot seem to find anywhere. Thank you in advance.
Neither. Do a viability stain as single cells are likely dead and clusters are viable. These are suspension cultures no need for a protease. Just aspire the entire or part of the media after a swirl, and put in a tube perform some light mechanical mixing by pipetting and centrifuge. Decant supernatant and add sterile PBS and mix the cells and centrifuge again. centrifugation speed should not exceed 1500rpm or 200xg depending on the roter you use. resuspend pellet in PBS, take an aliquot of a specific volume and add 0.4% trypan blue in equal amount to cell suspension. Or you can just take a sample from the culture directly and proceed with trypan blue staining. Incubate for 3 min at RT, then load a drop on a hemacytometer and view under a microscope. Dark blue are dead cells, do the count and see what you have (equation : % viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100 )
Thank you Rabeah for the detailed info.
Would it help to us typsin to get a single cell suspension? Or will mechanical mixing be fine?
Thank you again.
Simple mechanical mixing is fine especially since your cells haven't formed confluent clusters yet. Evan then mechanical is best, whereas trypsin may damage the cells stripping their surface proteins.
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