Hi, we are having trouble growing isolated mouse cortical astrocytes on coverslips for immmunofluorescence. We isolated the cells and expanded and froze some vials. After thawing, the astrocytes grow normally in plastic flasks, but when we plate them on coverslips for imaging many don't attach and the ones which do show an elongated spindle-like morphology.
For clarification, we are using poly-D-lysine (50 ug/mL) to coat our culture surfaces. Culture medium is high glucose DMEM + % FBS, 1X Pen/Strep, and 10 ng/mL each EGF and bFGF. We use media + 7.5 % DMSO for cryopreservation.
We have been treating our coverslips with 1 M HCl to hydrophilize them but no success. We have switched brands of poly-D-lysine, adjusted coating concentration, but no avail. We are still unsure whether it is a problem with the cells or the glass slips... So, wondering if anyone has experienced a similar situation, or has advice for seeding astrocytes on glass slips for immunostaining. Thanks in advance!
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