Counting MCF-7 cells - (Mar/02/2006 )

Hi,

I'm having some problems in culturing MCF-7 cells, they grow slowly (~ 1 week), and in clusters. When I try to count them is very difficult, because I can't count them properly.
I already tried longer incubation periods with trypsin/EDTA, pipetting up and down for ~ 20 times, and nothing, I still have a lot of clusters. How can I have single cells in a simple way? Should I add diferent enzymes to "dissolve" the clusters?
Can anyone help me?
Thanks

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Hi!

I usuallly culture MCF-7 cells in RPMI 1640 + 10% FCS and 100x PSF. That works well with them for me. To get rid of clusters you can put your tip of the pipet at the bottom of the flask and spit out the cells. Big clusters are now stucked to the bottom in your pipet and because of the pressure they will become smaller every time you re-do it. So pipetting up and down, but with your tip on the bottom of the flask.

Hope it helps!

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hi,
oh they grow so slow.... and they know exactly when you need them, so they take even longer .
but they shouldn't grow in clusters if you've been taking care of them. you shouldn't let them be overgrown, and (in my hands) they don't like to be split too harshly. when i let them get overgrown, they went clumpy... when i split them to harshly, they looked very sick.
have you tried syringing them with a 20 gauge (check this, i always get the measurements wrong, the big drawing up needle) at least 3 times.
if you've got a problem getting them off the plate, put in your usual amount of trypsin/EDTA and putting the flask back in the incubator for a few minutes. the heat helps.
you don't need to "dissolve" the clusters, just break them up. i recomment a syringe.

vetticus.

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Thanks vetticus3 for your reply.

I don't have problems in getting the cells out of the plate, they get out pretty easy. I already incubate the cells with the trypsin for a few moments before ressuspending them.
Also, the cells in the clusters seem fine, I just can't desagregate them, so I count with a huge error.
The cells grow in clusters right from the beggining, with low confluence. When they become confluent (70-80%), seems that they are not so much in clusters. So I think that they don't behave like yours: from what I understood, your cells only grow in clusters when they are confluent, right?
I've already tried to ressuspend more the cells (about 20 times), and they form smaller clusters. Next time, I will try with the syringe.

Thanks
Sorry for my english, hope that I didn't make any mistake!

pals

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hi,
your english is fine

if the the cells are only causing you problem with counting, definately try syringing them.

when i left my cells alone for too long, and they were too confluent, after i split them, they formed clusters very early. these clusters did not like trypsin, and did not lift off the plate too easily. the trick was to put them back in the incubator for a while.

best of luck,

vetticus

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