3 replies to this topic

[dfcomiskey]

Hi All,

I was wondering if anyone can offer any suggestions on how best to get MCF-7 cells or any other strongly adherent cells (HeLa S3) from sticking to one another after trypsinization.  Our lab works a lot with MCF-7 cells and when I am a splitting cells into treatment groups it can take FOREVER to get these cells into a single-cell suspension and frankly, my hands are getting tired from breaking them up so much.

We culture our MCF-7 cells in high glucose Gibco DMEM with 10% FBS, L-glutamine, and penicillin-streptomycin.  I trypsinize my cells using Cellgro 0.25% trypsin with 2.21mM EDTA.  To give you an idea of what I am doing: I aspirate the media, wash in half the media volume with sterile PBS (5mL for a 10cm plate), aspirate the PBS, then add 1mL trypsin (for a 10cm plate) and leave in the 37C incubator for NO MORE than 3 minutes, usually enough time for me to label and add media to a new plate.  I usually add 1mL of media to the trypsin and spend the next ten minutes with my 2mL serological pipet pressed up against the bottom of the dish to break these b*****rds up before diluting then even further with media before splitting.

I feel like adding FBS or something else to counter the effects of the trypsin wouldn't do much good because the damage is already done the moment I tap that plate and visualize them clinging to each other for dear life under the microscope.  Surely there must be a simpler way?  A better enzyme that digests ECM (hyaluronidase, anyone?) that would allow me to achieve a single-cell suspension more easily?

Thanks,

Dan

Edited by dfcomiskey, 17 April 2013 - 06:31 PM.

[cell slave]

Hello,
Yes, there is a really simple way, that works for me. Try using a syringe!!!

Here's what I do...

First of all, I wash the cells in D-PBS (2ml). Then, for a 25cm flask I add 2ml trypsin-EDTA and incubate at RT for about 30 sec. After that, I tap the flask vigorously on a clean surface and then add 5ml DMEM so as to inactivate trypsin. I add the suspension in a 15ml falcon tube and centrifuge them at 1250rpm for 5min. I discard the SP and add a few drops of DMEM in the tube so as to bring the volume up to about 1 - 1.5 ml.

Them I take an 1ml syringe (insulin syringe) and a 21G needle (green one, but please check cause in your country color labeling might differ).

I  pass the whole cell pellet through the syringe about 15-20 times. If I have one flask and want to split 1:3 I add more DMEM so as to bring the volume up to 3ml and then I add 1ml of the cell suspension in 3 new flasks and 4ml DMEM. I always get a single-cell suspension and viable cells.

Hope I helped!

[Tabaluga]

or perhaps with a microliter pipet (blue tip) ?

[dfcomiskey]

Thanks everyone for the tips!  I tried using a Luer-Lock syringe and a 20.5G needle and it worked beautifully!  So many hours of my life have been saved!

Edited by dfcomiskey, 21 April 2013 - 04:18 PM.