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Problems with HEK293 cells in 96 well plate format


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#1 Andras

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Posted 15 November 2009 - 04:44 AM

Dear All,
I've been having a little difficulty with 293 cells. They are not very adherent in the best of days (don't even require trypsin to dislodge them from the culture flasks), but it's been proving a disaster when I try to culture them in 96 well plates. We need them for RNAi experiments, so I really have to have equal amounts of cells in the wells. The problem is that at the washing steps, the ice-cold PBS simply washes patches off, no matter how careful I am. And I've been careful: adding the PBS dropwise onto the walls of the cells... it's taking a lot of time to do 20-30 samples, let me tell you :), and I'm still loosing cells.
(Strangely there's no such problem when I tried it on bigger plates; the only problem is, of course, that the reagents are a tad expensive to conduct the experiments in 6 well format.)
I thought about using EtOH or something similar to fix the cells onto the plastic, but I'm not really sure if it would work, and if it would interefere with the lysis, RNA isolation and qPCR. (I'm very new to cell cultures.)
I'm sure people use these cells in similar settings - please give me a few pointers what I could or should try.
Any help is much appreciated.

#2 amelia417

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Posted 15 November 2009 - 02:39 PM

Dear All,
I've been having a little difficulty with 293 cells. They are not very adherent in the best of days (don't even require trypsin to dislodge them from the culture flasks), but it's been proving a disaster when I try to culture them in 96 well plates. We need them for RNAi experiments, so I really have to have equal amounts of cells in the wells. The problem is that at the washing steps, the ice-cold PBS simply washes patches off, no matter how careful I am. And I've been careful: adding the PBS dropwise onto the walls of the cells... it's taking a lot of time to do 20-30 samples, let me tell you :), and I'm still loosing cells.
(Strangely there's no such problem when I tried it on bigger plates; the only problem is, of course, that the reagents are a tad expensive to conduct the experiments in 6 well format.)
I thought about using EtOH or something similar to fix the cells onto the plastic, but I'm not really sure if it would work, and if it would interefere with the lysis, RNA isolation and qPCR. (I'm very new to cell cultures.)
I'm sure people use these cells in similar settings - please give me a few pointers what I could or should try.
Any help is much appreciated.


Yes, we have the same problem with 293 cells. They are very sensitive to media changes and washes. For experiments where there are problems with adherence, we first coat the wells or plates with poly L-lysine (PLL). We use 2.5 mg/100 mL autoclaved water, and then filter after adding the PLL to the water. Add enough PLL to the wells/plates to completely cover the bottom and coat the plates for 1 hour. A longer period does not seem to help or have any adverse effects, but a minimum of 1 hour should be used. Simply aspirate the PLL and seed your cells into the plates. You do not need to rinse the wells, and should not do this in fact. We use Sigma's (full name is poly L-lysine hydrobromide, I believe). You can make a 4X stock in PBS (10 mg/100 mL), and then dilute this to 1X in the autoclaved water when you need it. Store the 1X and 4X solutions at 4 deg C. Be very careful with the stock PLL because it is very hygroscopic and has the consistency of cotton. It will turn into a big blob of goo if you get any liquid in the stock tube.

Edited by amelia417, 15 November 2009 - 02:40 PM.





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