Help with HeLa cells culture... - (Sep/19/2006 )

Hello,

Im really, really worried and need your help... I used to culture the cell line 3T3 and never had problems with contamination or anything.... BUT ! now, Im working with HeLa and SiHa lines... Something very strange have been happening to me...
I put my cells in my plate one day, and the next morning, all of them are floating dead in the medium !!!!!!! Is horrible !!!!!

It is not contamination...
The medium color is normal...
They just die ! I have no idea what I do !

I use DMEM medium, with 2% serum and 1% antibiotics... I use trypsin... centrifuge at 2,500 rpm....


Do you have any clue ??? Do you think it is possible that I manipulate them too strong so I harm they and kill them ? I mean, like when I put them in the plate or anything like that ???

Am I using too much antibiotics ???

Please help me !!! Im so sad and stressed because I have no idea what am i doing wrong !

Try using 10% serum instead. Btw, what's ur Trypsin concentration? Too much Trypsin is harmful to the cells...........use the minimal amount just to cover the layer when detaching.

If the above method is not working..................get a new batch of cells. Probably, ur current cell line has undergone many passages.

Good luck............

I cultivated HeLa cells with DMEM + 10% FBS and 1% Pen/Strep. The centrifugation could be a bit too strong, try with 1500 rpm for 2min

Hela's should be the easiest cells in the world to grow.

RPMI or DMEM (1000mg/L Glucose) + 10% FCS/FBS + 2mM Glutamine NO NEED FOR ANTIBIOTICS if your technique is good enough.

Always use 0.25% Trypsin OR LOWER

100g for 3 minutes is easily fast enough and long enough to spin cells. RPM means nothing and remember that centrifugation DAMAGES CELLS.

I would agree with others on using 10% FBS and spinning them at a lower speed.

Just a stupid question, r u using a new batch of FBS/FCS?

hi la celulita,

How do you store your cells ?? It looks to me that the cells are already dead, so they are not correctly stored

I also use DMEM + 10% FBS and 1% Pen/Strep for my HeLa cells,
and I centrifuge at 2100rpm

I think you should order some new cells,

good luck

I usually centrifuge cell for 5 min. at 1000 rpm. I don't know what is the equivalent of the rpm's in g's.

Hi LaCelulita,

As Rhombus said Hela are the easiest cells to culture. You might want to consider following factors:

Reduce your centrifugation speed, we generally use 1000 to 1500 rpm for 5 to 10 min.
2% serum is too low, try 10% serum,
check whether there is something wrong with the serum batch.
Check the actual temperature in your incubator.
Are you using right tissue culture flask for adherent cells? Just check whether they are hydrophobic flask made for suspension culture.
Which antibiotic you are using? Check the final recommended concentration of the antibiotic.

You can also suspect the cell stock itself.

Hope it helps.

Hello Everybody !!!

Thank you so, so much for all your responses and help.

I put new cells today with all your recommendations about centrifugation, no trypsin, I used versen plus I used the antibiotics at 0.5%...
The only thing I couldnt do was using the FBS at 10% because I need to grow my cells actually with 0.05% of FBS... so, 2% is a luxury at this moment....

I have a question about centrifugation though... where do you centrifugate ? I mean, I use 1.5ml eppendorfs.... So, should I use a lower speed ? Thank you !

Wish me luck !

hi
they meant i think that the routine culture for having helas in healthy way is to do in 10% serum. Switch to other percentage only for your exp, but do a good stock before in case you need to defrost again.

I do culture on 10cm or 1cm plates so spins are done in 15ml or 50 ml tubes, at 200-300g.