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