Can someone recommend a simple protocol for preparing HeLa culture? Or can you give some details?
Hello. First you should confirm the medium specifications from the Seller as different sources may specify different medium make up but basically the 10% FBS and 1X penicilin+Streptomycin should be okay. Then U should thaw the cells in the water bath at 37C and remove/discard the storage solution (FBS+DMSO). Then suspend the cells in about 4ml medium and seed in 60mm dish (Check the cells under the microscope to have an idea of the density and confirm that the morphology is that of HeLa cells!). Incubate at 37C in a 5%CO2 humidified Incubator for 24h. After a day, change the medium as some dead cells may be present. Revitalize with new medium and check under the microscope again. Keep cells in the incubator and after 24h, the dish should have reached up to about 90-100% confluence. You should then passage into a 100mm dish.
In passaging, you must take all the necessary precautions. (Warm d medium, trypsin and PBS in d water bath at 37C)
-Check cells under the microscope for density and morphology
-Remove the old medium from the adherent cells
-Wash with 1X PBS
-Digest with 1ml of trypsin
-Centrifuge at 800rpm for 3min
-Suspend cells in fresh medium (about 4ml)
_Then do split passaging depending on how soon U want to use d cells.
_Check cells under the Microscope and keep in the incubator.
P.S. Sometimes, the cells may trick up, having funny morphologies or not even growing properly. I guess U will need to do the passaging under the supervision of someone who has experience. it's important. I had series of problems with my HeLa cells too.
All the best!
Thanks Adeola for your deep reply, it realy helps. please could you recommend or upload some references.
Hi Ashraf Ali. Sorry for d late response.
Some links below for your use.
http://helacells.blogspot.com/search/label/Hela%20cell%20culture%20protocol%20hela%20cell%20doubling%20time
http://www.abcam.com/ps/pdf/protocols/cell_culture.pdf (This is a general guideline but u'll find it useful, I did.)
All d best.
P.S. I forgot somehow to mention that t DMEM makes the remaining 89% volume of the culture medium.
I am so appreciated for yor deep and details answer. and thanks for the provided texts. It realy helps.
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