I digested brain tissue with 0.25% trypsin at 37°C for 30 minutes, then isolated microglial cells using 30% Percoll gradient centrifugation. I cultured the cells in 5% MEM or DMEM medium, but the cells barely adhered. Under the microscope, the adherent cells appear as small, round dots and lack the typical morphology of microglial cells. I also tried coating the plates with P-L-L, there was no improvement. I confirmed the cell phenotype using flow cytometry, identifying them as CD11b+CD45int and CD11c+. What could be the reason?
If they are barely adherent, did you try growing them longer? Also, are you using any complete media, serums (Heat inactivated or no), any hormones, sugar or amino acids that might be necessary? Also different brain tissues from different species need different types of media to grow and/or adhere.
Andreas Michael Thanks for your help. I cultured the cells in vitro for nearly a week, but the adherent cells appeared more like astrocytes. I used MEM and DMEM media, which already contained glucose and L-glutamine, so I only added heat-inactivated FBS and antibiotics. I have tried once adding glucose to the MEM medium, but the cell adherence did not improve. My microglial cell isolation and purification procedure, as well as the culture medium used, were completely based on published literature. However, the cells just won’t adhere. I’m really perplexed.
Hello Echo Zhu,
The use of high trypsin concentration may have significantly reduced the cell's ability to form adhesive bonds with adsorbed cell adhesion proteins by decreasing the number of functional integrins available on the cell membrane.
You may prevent integrin damage by using either low trypsin concentration or reducing digestion time from 30 minutes to 10-15 minutes with periodic shaking, which may result in substantially improved cell adhesion.
Best.
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