Please help! harvesting fibroblasts (by centrifugation from culture) - (Oct/03/2021 )
I'm new to working with fibroblasts. I'm growing them in T25 cell culture flasks (in 5 mL media) and I need to harvest them for protein analysis. I've tried twice and my harvesting efficiency is extremely low. It's driving me a little nuts that the pellet is invisible and I know that I'm discarding most of my cells in the supernatant, but I don't know how to prevent it/improve it.
My centrifuge is this one: https://www.socalbiomed.com/catalog/product/view/id/7842/s/waverly-cl4m-mini-clinical-centrifuge/category/22/
it can go from 300 to 5,000 rpm
The protocol that I've been attempting to follow calls for centrifugation at 100xg for 10 min (4x) at 4 degrees C.
This protocol also involves 3 washes in PBS or HBSS to remove the media/trypsin before freezing the cell pellet for later analysis
However, we don't have a refrigerated centrifuge, so I tried following this protocol at room temp.
I assume that this temperature discrepancy may be the issue if the cells form a tighter pellet when chilled, but I've read online that some people harvest fibroblasts at room temperature. And the centrifugation speed seems to vary widely among protocols.
So I'm hoping that someone can suggest a protocol that is suited to my equipment.
Although I'd love to hear anyone's protocol...even if it means having to purchase new equipment.
Essentially, I have fibroblasts growing on the surface of a flask, and I'm looking for protocols through which I can get those cells into a "clean" cell pellet for protein analysis. I can purchase different types of consumables. I could even attempt to find a means of refrigerating the centrifuge if absolutely necessary. But money is tight so I'm hoping to find a protocol adjustment that's as inexpensive as possible.
Can you add details of your protocol please. We need to know what you are doing so that we can work out where you might be going wrong. We will need to know the major steps and reagents used. For example it might look like this:
- wash cells in PBS 3x.
- Lyse in XX ul of Y lysis buffer (z% SDS, 0.1 mM deoxycholate, 0.9% NaCl, 1x protease inhibitors).
- Pellet cell debris at R RCF for T min.
- Freeze.