Hi Tim most cells don't like to be plated at a very low cell concentration. They like to have a certain number of siblings around. Cells often make various growth/survival factors. When the cell number is to low the amount of these factors to stimulate growth/survival drop below some threshold and the cells often just "sit" until enough of the factors accumulate and the cells will begin to grow.

Here's a tip I often use to keep single cells happy. Take medium from a 48-72 hr old MCF7 culture and sterile filter (0.22 micron). Use medium from sub-confluent cultures only. Add this conditioned medium to fresh medium (30% conditioned/70% fresh) and us to feed your cells. This normally supplies the survival/growth factors the single cells require and they should make colonies faster.

Cell culture ON!

Dan

In 96-well plates, diluted to about 1 cell per well, you should get an amount that can be transferred to larger plates (12 or 24-well) in a bit more than a week. Doubling time for this cell line is about 24h, depending on culture conditions of course.

Hi Tim most cells don't like to be plated at a very low cell concentration. They like to have a certain number of siblings around. Cells often make various growth/survival factors. When the cell number is to low the amount of these factors to stimulate growth/survival drop below some threshold and the cells often just "sit" until enough of the factors accumulate and the cells will begin to grow.

Here's a tip I often use to keep single cells happy. Take medium from a 48-72 hr old MCF7 culture and sterile filter (0.22 micron). Use medium from sub-confluent cultures only. Add this conditioned medium to fresh medium (30% conditioned/70% fresh) and us to feed your cells. This normally supplies the survival/growth factors the single cells require and they should make colonies faster.

Cell culture ON!

Dan

thank you both, I'll definitely try that Dan! Now they are indeed just 'sitting'. They don't die, but they dont grow either. I'll let you know if it worked

I've used this single-cell-in-96-well method for making clones of transfected MCF-7, with succes. You could try to add some extra FCS, to 15% instead of the usual 10%, that may help. Serum batches vary a lot in quality, thereby strongly affecting colony forming efficiency.

I will try different methods on different cells. I will increase my FBS to 15% on 1 cell, use 30/70 medium on 1 other and perhaps combine the 2 on another cell to see if the cells will do something. Do you use a certain probe or dye to follow cell growth or just a normal microscope

Turned out that RPMI-1640 made from powder(without phenol red) works better then normal medium. It took 2days for the cells to form a clump and around a week until a reasonably sized clump had formed. The first day they were in 15% FBS after that normal medium

Fine it worked out, then. Cells are sometimes fussy about the medium indeed. I don't know if this is the case for you, but RPMI traditionally contains glutamin instead of glutamax. The glutamin eventually degrades to ammonia, which is toxic. Serum also needs to be relatively freshly thawed, and quality differences between batches/suppliers are considerable (see also the problems with PAA, you might have heard from that).

I ended up using RPMI-1640 supplemented with 10% FBS pen-strep, L-Glutamine and 0.1% bovine insulin on cells with passagenumber of 16

do you succeed to grow up single cell mcf7 ? previously I used High glucose DMEM + 10% FBS and condition medium but after one week my cell did not grow up

I use 10% FBS + high glucose DMEM for my MCF7 cells and they grow fine, whether it is regular passaging or cell isolation.

Its a while since you asked, but I'll put in my tuppence worth anyway.

I used to do clonogenic survival assays using MCF-7 cells. I would seed 2000 cells per plate and expect to see colonies of 50 cells or more at around 12 days. The plating efficiency was around 50% in control plates. I was using High glucose DMEM, with L-glutamine but without sodium pyruvate and 10%FBS in 10%CO2. I had similar results growing them in Iscoves modified Dulbeccos medium/10%FBS at 5%CO2.

Hi, how often do you change media at the MCF-7 colonies after sorting?

Thanks