Cell culture and live cell imaging

I am growing the HEK-293T and HeLa cells for live cell imaging purpose. They are perfectly adhered and growing in T-25 flask and plastic 6-well plate. However I am having the problem of adhering these cells with glass surface. They are not adhering to cover slip or glass bottom plates even after coating the glass bottom wells with Poly-D-lysine. Could you please help me out how can I fix this problem? Your suggestions will be highly appreciated.

Hey mahmad, what coverslips / glass bottom plates are you using? What media are you using when transferring your cells to glass? HEK can sometimes be a bit tricky but there is no good reason that HeLa should not attach, unless they are unhappy/dying.

Hi Andrew, thanks for your reply. I am using 1.5 mm coverslips and 6 well glass bottom plates (cellvis, USA). Advance DMEM (10%FBS), It’s same media we use for ruotine cell culture. If they are unhappy, should not grow in flask as well. However they are adherent to flask and 6 well plate (polystyrene).

That’s very odd then that they won’t stick, even with poly-lysine. Maybe the Cellvis plate has something on it that is making the cells unhappy? Do the cells adhere when you put them in a 6 well polystyrene plate with a #1.5 coverslip inside. It wasn’t clear if you used the coverlip inside a cellvis plate or just a normal polystyrene plate.

Alternatively, you could try and use a polymer coverslip like Ibidi. It has properties similar to a #1.5 coverslip. Just be careful what oil you use as some of them damage the polymer.

Also there are the Labtek and Matek glass options that you could try.

I did in both way, by putting the coverslip in a polystyrene plate and seeding the cells on it. Cellvis plates are already glass bottom (#1.5). These cells are showing the unusual behaviour when they come in contact to the glass surface not on the polystyrene surface.I also tried by coating the glass surface with poly-d-lysine, but did not help

I agree with @Andrew_Seeber that this is unexpected, especially for the HeLa cells. A few additional questions to make sure we are all starting from the same strong foundation.
Did you try uncoated glass? I think you did, but want to check. HeLa’s are usually fine on uncoated glass.
How long after plating the cells are you viewing them? It’s possible it might take a little bit longer for them to spread out after plating on glass than on the TC coated plastic. If you’re looking at 4-6 hours I wouldn’t be shocked if the cells on glass are barely adhered. If it’s overnight, there’s definitely something strange going on.

An alternate strategy to Poly-lysine that I’ve used is to incubate the coverslips in complete media overnight before plating the cells (this allows time for the proteins, etc. from the serum and media to adhere to the glass and generate a hopefully more adhesive surface). I did this by setting up my culture plates plates the night before doing the cell split instead of during the trypsin incubation.

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This is very unusual for me too. As suggested by you, I will give a try to incubate them in complete media O/N before splitting the cells.

Thanks

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I also find it useful to treat the coverslips before I add cells to them. I have done Piranha wash (H2O2 + Sulfuric acid), plasma cleaning, and EtOH + flame to remove the hydrophobic coating before plating cells. Flaming coverslips after dipping them in EtOH, washing them in diH2O, and then UV sterilizing them is the easiest and generates the least toxic waste. The biggest caveat is to be sure to allow most of the EtOH to drip off of the coverslip before introducing it to the flame and to then move the coverslip around after it’s aflame to avoid having any one region superheat and crack (be very careful with dropping flaming EtOH into the EtOH container). Treating the coverslips like this will improve the spreading of an adhesive like PDL or conA, and HeLa cells also prefer to bind to glass where the coating has been removed. I also find it always useful to give the cells at least 24 hours before fixation.

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Hey Mahmad,
Ever checked “adhesion slides” (https://www.marienfeld-superior.com/adhesion-slides.html -> no commercial interest). EVERYTHING sticks to those slides - I have used them for years for suspension cells - always works. I have no clue what kind of coating they use, but they really work well and allow ease of staining as well. Good luck.
Josef

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These look awesome and I had never heard of them before. Thank you!

Mahmad, Any luck getting the cells to stick?

Hi FishCsCells, Thanks for checking. I tried by coating the surface with collagen and now I am getting 80-90% adhesion to the surface.

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Thanks for sending the link. I will check them out.