Hi Tzachy.

Add equal vol of 4 mg BSA/ml PBS (PBS+BSA) to the cell suspension that has undergone stopping the enzymatic digestion with FBS, followed by washing the enzymes and serum away in PBS.

Mix the cell suspension and PBS+BSA gently with pipette to make final concentration of 2 mg BSA/ml PBS.

Avoid hard spinning down which makes cell aggregates due to sticky surfaces after the digestion.

Use the 2 mg BSA/ml PBS throughout the cell labelling, washing and sorting.

Best luck.

Hi Tzachy.

Add equal vol of 4 mg BSA/ml PBS (PBS+BSA) to the cell suspension that has undergone stopping the enzymatic digestion with FBS, followed by washing the enzymes and serum away in PBS.

Mix the cell suspension and PBS+BSA gently with pipette to make final concentration of 2 mg BSA/ml PBS.

Avoid hard spinning down which makes cell aggregates due to sticky surfaces after the digestion.

Use the 2 mg BSA/ml PBS throughout the cell labelling, washing and sorting.

Best luck.

First wash your cells with pre-warmed PBS contains 19 mg EDTA.4 Na/100 ml of it for three times. Then , warm up Trypsin-EDTA. Additionally, centrifuge at 350g for no more than 5 min. Then they would be separate by gentle pipetting.

you must optimize the time and the speed of centrifugation for collection of cells, you may used more time or high speed. Additionally, you must good resuspend the cell aggregate.

Dear Tzachy

They are some really suggestions given above by different experts. I think we are missing one obvious point here. Why are your MSCs making such clumps? They don't do that often and it is easy to make single cell suspension by pipetting. Might be your cells are getting too much confluent and thus form a monolayer sheet which forms clumps when detached and centrifuged. It generally happens with corneal stromal stem cells and fibroblasts. I would recommend you to take care of that. Kind of very unexpected but hope you are also not using ethanol at any step for fixation or anything.

Generally in our lab, we use FACS tubes with filter caps (blue caps). We pass the cells through the filtered cap while transferring in FACS tube. That get rid of any clumps. You can try that if vortex and pipetting are not helpful.

Hope it helps!

Thank you all for your answers.

Sang Ho Lee, you mean to use the PBS/FBS solution to stop the enzymatic process and also as a washing buffer (in different ratios of course)?

Farjam Goudarzi, I didn't understood what did you mean by " 19 mg EDTA.4 Na/100 ml " ?

Ajay Kumar , I use placenta stem cells but of course they are creating mono layer on the flask, this is what I want, how else will I will increase the number of cells? how can I take care of that? what do you do differently?

I dont use ethanol in any stage and I'll try using those FACS tubes.

Thank you all for your help

I meant 19 mg edta per 100 ml pbs then autoclave or filter. Remember first dussolve etda in NaOH thwn add to pbs.

Thanks for the fast reply, we will try

Dear Tzachy,

Cell clumping usually occur when there are many death cells that release the DNA.

To avoid cell clumping, you have to detach gently, use TrypLE Select instead of Trypsin, neutralize with equal

volume complete medium (containing serum or platelet lysate, as serum neutralize the enzyme)

Pippette carefully to make single cell suspension and when clumping still occurs, you can add DNAseI to prevent

cell clumping.

You can read the method in this link:

https://www.stemcell.com/dnase-i-treatment-for-clumpy-cell-suspensions.html

Good luck

Hi Tzachy.

Yes. PBS + FBS as a stopping buffer, and then PBS + 4 mg BSA/ml as a washing buffer to save expensive serum. Keep the tube and fluids on ice, too.

Best wishes.

Dear Tzachy

Formation of a monolayer is definitely required and its property of cells. But overconfluence leads to formation of a really tight sheet which gets cluster together after trpsinization and cells are really difficult to separate apart then. So take care that you are not letting cells to be 100% confluent. Harvest them at 70-80% confluence so that cells don't stick with each other. And the tubes I suggested can definitely help.

Hope it helps!

I'll second the overconfluent cells comment made by Ajay Kumar. If you allow the cells to become too confluent before you passage them there will be a lot of ECM proteins present that cause aggregation and clumping. Try to split your cells at about 75% confluence. If they get too confluent, a pre-digest with collagenase type I followed by trypsin generally works. You could also try a serum free cell culture media, as cells grown without serum are a lot easier to detach anc process.

Ajay and Brenton are right - grow your cells to about the confluency shown in Fig 9.3d of attached and you'll have no issues. BTW, you get the best cells by far for experiments if you only culture to this confluency.