Hi,
I'm doing a chemotaxis assay, and need to coat 24 well plates with Polyhema to prevent my neutrophils from sticking.
I seem to be having a problem when I come to use the plate for the assay, whereby the wells become 'smudgy' - it looks like the polyhema is coming out of the solution. Initially I thought I was making too large a volume in one go, and thus the ethanol wasn't getting warm enough to dissolve the solution. By doing several smaller volumes. This worked fine intially, but today I found that, although my wells were clear coming out of the incubator (I incubate for 24hrs) once I added media to wash, the wells became smudgy again.
Any advice?
Hi Sophie
McCaffrey Lab - Polyhema coating tissue culture plates
Low adhesion dishes – coating with Poly-HEMA (Poly 2-hydroxyethyl methacrylate;
Sigma Cat # P3932)
1. Dissolve 2 g in 100 ml 95% EtOH by rotating several hours @ 65 C. This gives a
20mg/mL solution.
2. Coat plates with 4 ml/10 cm plate. For 6-well plates or 35 mm dishes, use 700
ul/well.
3. Dry overnight @ 37 C. Plates can be wrapped in parafilm and stored indefinitely
at room temperature prior to use.
To prepare, we use the same method as Awatif. However, to coat I use 1ml for 6 well plate and let it dry in the laminar flow hood O/N to keep it sterile.
Good luck
Hi both
thanks for the tips> I seem to have created a way that helps - preheat the ethanol at 50 degrees, then add this to the polyhema. alternative vortexing and heating until dissolved then add to a warmed plate, and put in the incubator. I think the issue before was that a) the ethanol wasn't warm enough, b) I was dissolving one large volume instead of several smaller volumes and c) the plate and media when I wash before use wasn't warm enough
I have the same issue, but I am adding only small volumes and vortexing the poly-HEMA + ethanol dilution for 12 hours.
The only thing that I do not do is to heat the poly-HEMA as I am using one with very low molecular weight compared to the Sigma Aldrich one (Sigma: 300 kDa, the one I am using: 26 kDa). Does someone know what could be the possible reason?
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