PLL and Laminin Coating for primary rat cortical neurons - Cell Cluster and die (Sep/26/2007 )
Hi all,
I hope someone can help me on my cell clustering problem.
What I did was to simply add 500uL of PLL+laminin solution (containing 33ug/mL poly-l-lysine and 2ug/mL laminin, both in water) to 6 wells plates for 2hrs in the 37 incubator, rinsed with water (2x) prior to plating the cells. However, neurons aggregated 3 days later and died subsequently.
Please help!
Thanks in advance
I hope someone can help me on my cell clustering problem.
What I did was to simply add 500uL of PLL+laminin solution (containing 33ug/mL poly-l-lysine and 2ug/mL laminin, both in water) to 6 wells plates for 2hrs in the 37 incubator, rinsed with water (2x) prior to plating the cells. However, neurons aggregated 3 days later and died subsequently.
Please help!
Thanks in advance
We coat with poly-lysine O/N and then with laminin for 1 hr RT.
Also have you checked if the cells are evenly distributed and are single cells when you plate them. If not this could also be a problem. try plating them at higher densities.
good luck !!!
I`ve had the same problem. We work with primary hippocampal neurons from E18 rats. We used to icubate acid-treated coverslips in 500uL of poly-L-lisine (10ug/mL) overnight at the CO2 incubator, and then wash three times with water, and it worked well. Suddenly The cells started to agregate after one or two days in culture (they were completely dissociated after plating), forming thick fasciculated processes after some days. What we did was increasing the concentration of the PLL solution to 100ug/ml or 1mg/ml and the problem was completely solved!
Sometimes PLL gets hidrated in stck and you may have biased weight. Test higher concentrations and make sure your coverslips are perfectly acid-treated (overnight in 70-100% HNO3), and not stored for too long.
Sofia.