I'm unable to currently continue the work on my thesis due to losing a huge number of cells in wash steps (up to 75% after 2 washes).
I'm using 2 cell lines Kasumi-1 and Molm-14.
I'm working in 1.5ml microcentrifuge tubes and washing with 1x PBS. The cells are not being burst as I can see them if I save the supernatant and run it through the cytometer.
I was previously able to pellet the cells for washing after fixation, but in a swinging bucket centrifuge at a collaborator's lab which I do not have access to on a daily basis.
I have currently tried 200g for 3, 5 , 10, 15 and 20 minutes; 400g for 5, 10, 15, 20 minutes; 500g for 5 minutes; 800g for 5, 10, 15 , 20 minutes; 2000g for 5, 10 minutes. Some conditions provided slightly better or worse results, but none were good enough to move on with my full protocol, which requires pelleting and resuspending the cells ~10 times.
I have also tried adding 0.5% BSA, but it did not seem to help and disrupted the counting of cells in the cytometer.
This is my first project working with eukaryotic cells so I am a complete beginner, so any advice would be appreciated. I have deadlines approaching and am at my wits end at this point.
From what you describe, are you just fixing cells and then washing the fix out with PBS prior to then running them on a flow cytometer? If yes, then:
1. What is the volume of fix are you using and how much PBS for washing?
2. 300xg in a microfuge for 5 minutes should be adequate to pellet the cells.
3. Why are you washing 10 times?
Hi Tom,
Thanks for taking the time to respond.
Right now I have just been running tests with previously fixed cells without carrying out my full protocol to attempt to optimize the centrifugation.
1. . The cells were fixed with 10 ml 4% formaldehyde for 20 minutes and then washed twice with 50 ml PBS, before final resuspension in 30 ml PBS.
2. I know 300xg for 5 minutes should be enough but it is not working.
3. In the real protocol I will be permeabilizing cells and then adding different enzymes in individual steps to complete different reactions (can't really get more specific because I'm under an NDA).
So, the 10 washes are washes after each individual step to remove the buffer from the previous step.
Thanks for the information.
Have you checked to make sure the centrifuge is actually spinning at the correct speed/tried using a different centrifuge?
How many cells are/will you dealing with?
If you can do everything in small volumes/Eppendorf tubes (i.e. 100ul fix then 1ml PBS wash) you will be better off than using larger volumes.
If you have to use larger volumes you should minimize the volumes as much as possible (i.e. 1ml fix then 5-10ml PBS wash). Cells spin out better in small volumes.
It's inevitable that you are going to lose cells the more washes/spins you do. So in that case you want to start with as many cells as you can such that you end up with enough at the end for analysis.
The centrifuge is a brand new Fischerbrand accuspin so it should work. I have not tried using a different centrifuge as it is the only suitable one available in the lab.
In my current optimization of the centrifugation I have been running with about 5k cells per tube and ending up with between 300-1000 cells after 2 washes. In the full protocol I started with 1 million cells and ended up with only hundreds of events at the end of all the steps.
The experiments I'm currently running are performed with 1 ml PBS for washing and resuspension.
To update, the issue has basically been resolved.
The primary problem was seemingly the freshness of the cells used. Cells were much easier to pellet on the day of their fixation. After storing the fixed cells for only 2 days (over the weekend), they were much harder to pellet compared to the day of fixation.
Ended up using 800xg for 5 minutes for centrifugation.
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