Hi everyone :)
I work with SCC-7 Cell lines. The medium is RPMI, 10% FBS, and 0,1% Strep-Pen.
They grow well in 100pie dish during thawing and subculture.
But when I start to seed them in 96 wells plate, they do attach to the bottom, but do not show any dendrites, some of them undergoing apoptosis and some of them start to grow in colonies (picture attached).
I already clean all the clean bench and incubator. Try to put PBS in the blank edge wells to reduce the medium evaporation. Seed even less and more cells ( 5000 cells/well and 10000 cells/well). But it seems all of them did not work.
I would really appreciate all your suggestion :)
thank you
Best regrads.
Hi!
Do you use 96-well plate and petri dish from the same plastic? (I mean the same company and the very same material.) Some cells just don't grow on any plastic even if they are coated with gelatin/poly-L-Lys etc.
Good luck!
Hello Carolina
I would suggest you to use some attaching matrix in 96 well plates instead of plating them on plastic. As far as I know, Collagen and laminin works good for attachment and also help in the process of dendrite formation.
Hope it helps.
Thank you so much Mr. Cervenak , Ms. Kaur, and Mr. Ikink. :)
I just checked the plastic ware. It seems to me that they are from the same type. They said it cell cultured treated polystirene.
(H11100) 100pie dish http://www.hdmicro.co.kr/eng/product_kr/p1_plas_03.php
(H30196) 96 wells plate http://www.hdmicro.co.kr/eng/product_kr/p1_plas_05.php
I will try to incubate the plate with 10%FCS before seed them this time.
Dear Ms. Kaur. Would you like to explain me more about the collagen as attaching matrix? I never use attaching matrix before, a bit of step how to you use collagen will really help ^^.
Thank you so much!
I don't have experience with the SCC-7 cells, but overall there are several things that may play a role. Why do you want a growth of these cells?
You already mention filling wells around the edge with PBS to help with evapouration, but this is definitely an issue with 96 well plates - perhaps a property of their surface area/volume ratio, or to do with the amount of media added.
Would definitely recommend growing in an incubator with controlled humidity, filling all empty wells (not just edge wells) with PBS (or maybe even with media) and trying to have >250ul media per well for the cells. Topping up the media every day or two might help; if the cells are growing quickly, they might be rapidly depleting the available serum (I know it should work to scale down from a 10cm plate, but I sometimes find that balancing cell number to well size is a matter of trial and error.)
how long time you take during preparing cells (trypsinization, washing, counting, and seeding in 96 well plate)? some cell lines are sensitive if you spend long time in this part. try to reduce the time of trypsinization to keep you cells healthier. are you using round bottom or flat bottom 96 well plates? round bottom plates may give the results that you observed
I agree with the view of Mr. Wamidh H Talib that some cell lines are sensitive if you spend long time and you should reduce the time of trypsinization to keep your cells healthier. I know that round bottom plates may give the results that you observed.
Thank you so much for all suggestion and help !! ;)
I really appreciate that.
I tried to adjust the volume each well and coat with collagen and it do help. Thank you so much!!.
I also fill all the wells to reduce the evaporation and speed up the step during counting and reduce the time during trypsinize.
I did all the suggestion in the same time and it help my cells to grow well in 96 wells plate.
thank you so much for all the experts. All the best for all of us.
Best regard. :)
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