Hi, I am conducting experiments with mammalian cells, for now 3T3s.
I wanted to wash the cells after coating in polyelectrolyte aqueous solutions, and, as I have always done, I centrifuged down the cells with 1000 rpm, for 5 min. But I found that a significant loss of the cells after discarding supernantant compared to the cells in cell culture media.
Do you have an idea of how to wash the cells without using centrifugation?
Thanks in advance.
Losing cells during centrifugations and washes depends on the type of tubes or plates you are using. If you are using flat bottom plates (96-well), the loss will be more. If you are using round bottom plates, the loss is a bit less. V-bottom plates are best for washings.
However, in your experience losing cells with the aqueous solution but not with the culture media is due to the presence of serum mostly. The presence of serum promotes the formation of a tight and sticky pellet which can with stand shear forces during removing the supernatant. In my experience, adding serum to the PBS before washing prevented the cell loss.
If you really have to avoid adding serum in your experiments, it is better to use V-bottom plates or tubes. I normally leave some supernatant above the pellet to make sure not to touch the pellet. I hope this helps you. Goodluck
3T3 are an adherent culture - why is centrifugation necessary at all? Simply aspirate the covering, add PBS to wash, swish it around gently if you like, and aspirate it away. You should lose only loosely attached/dying cells.
Of course I know I don't need extra washing steps if I want to just mantain the cells or do some experiments with the cells after seeding on plates. Washing is essential for this experiment. I am trying to harvest the cells and modify the cell surfaces with some materials I synthesized.