I'm growing RAW 264.7 cells on poly-l-lysine coated cover slips in 6 well plates, infecting them with bacteria, then doing May Grunwald Giemsa staining on them. Following this, they're being mounted onto slides with DPX. This obviously requires a lot of wash steps. The RAW cells attach to the cover slips just fine, but the cover slips themselves keep sliding around the wells whenever I move the plate or wash them. Anyone have a suggestion for a good way to get them to stay in one place that won't interfere with staining/removal/mounting/sterility?
Hi!
You can use smaller wells to reduce moving coverslips if it is possible. if volume of the well is critical, you can take a minimal volume of agar, drop on the bottom and fix the coverslips. Be careful: surfase tension could break the coverslip. Drop agar closer to one of its conners. Note: do it under aseptic conditions!
Good luck!
Hello Alexandra.
As Tatiana answered, use 24-well dish. However, you need to take out the coverslip carefully at the final stage. It is best to do washing and staining procedure in the air by putting your coverslips on a tub of 1.5ml Eppen-tube (cut-off the lid) attached (using super-glue) to the bottom of transparent box with a lid containing shallow water for humidity. There is no way to avoid sliding in your well since bacteria living under side of the coverslips provide gap between bottom of the well and coverslip, allowing fluid flow underside of the coverslip.
Good luck.
Tatiana: Unfortunately we only have a couple 24 well plates left (but a ton of 6 well plates) and I don't believe we have any cover slips that can fit in the wells on hand, so I'm sort of stuck with the 6 well plates. For the agar method, is using plain bacto agar fine because it's such a small amount? Or does it need to be noble agar or agarose to avoid upsetting the cells?
Hi Alexandra!
Dependently of agar characteristics (viscosity) you have to take 5 or significantly less ul agar. Its quality doesn't metter since it doesn't contact cells. It have to be enough for preventing coverslips sliding but have to allow their easy manipulation. If you don't need in coverslip moving from the dish you can use special petri dishes with glass bottom. http://liveassay.com/catalogue-2/35mm-dishes/
Good luck!
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