Good day to all,
I have been having trouble plating my HeLa cells into Ibidi 8-well chambers for confocal imaging.
My HeLa cell stocks have been growing fine in T-75 flasks, however, they do not seem to adhere to the Ibidi 8-well chambers.
Typically, I use the same protocol;
1) Coat ibidi 8-well chambers with 200 uL Poly-L-Lysine solution and leave for 5 min
2) Aspirate Poly-L-Lysine solution, wash each well once with MilliQ water, and leave in fume hood to dry for ~2 hr.
2) Add 10mL DPBS into cell stock. Aspirate.
2) Add 2mL Tyrpsin-EDTA (1X) and incubate for 2-3 min in the incubator.
3) Add 8 mL DMEM medium (with P/S & 10% FBS) and transfer to a 15 mL Falcon tube
4) Spin Falcon tube, remove supernatant and resuspend in DMEM medium
5) Count cells and plate 11x10^4 cells/mL into each well
Tiny dots of HeLa cells can be observed after plating. However, after 2-3 days, the cells don't seem to adhere and still appear rounded.
I have been trying this for nearly 2 months to no success. And I have been very delicate plating the cells, seeding them one drop at a time.
Please assist if you are able to.
Much appreciated.
Warmest regards,
Lewis Tan
I'm not famiiar with the Ibidi chambers, but I presume these are glass bottomed. Like many cells, HeLa's don't do as well on glass compared to plastic (like flasks, where they grow like gangbusters). If the splitting procedure you use on the chambers is similar to the T-75 flasks, then the issue is the glass coverslip. 5 min of poly-lysine on glass is far too short in our experience. We typically coat glass overnight at 37˚C, or for a minimum of 2 hr. If not that, try remaking your poly-lysine, as it goes bad (if you are not using frozen aliquots; you should). Polylysine should be higher molecular weights (like >70kD) and should be ~.1ug/ml concentrations. Good luck.
Good day Bryen,
Firstly, thank you for your reply. Secondly, the Ibidi chambers I am using are not glass bottomed. Attached is the information sheet from Ibidi and it actually stated that the chambers are plastic with a polymer coverslip on the bottom. Additionally, I have coated the slides with Poly-L-Lysine solution (Sigma-Aldrich) as stated from the protocol on their website. As a result, I am stumped as to why my HeLa cells have not been adhering to the chambers and even if they are, they do not appear HeLa-like wherein they appear rounded (see attached pictures). I tried replacing the DMEM medium the next day but they still do not adhere the following days.
So if anyone has any ideas, it would be much appreciated.
Warmest regards,
Lewis Tan (18768730)
Hi Lewis, according to your experiment procedure, there is no principled mistakes. I was not used Ibidi 8-Well Chamber, but I have made confocal images with Millicell EZ SLIDE 8-well glass( cat no.PEZGS0816) which works well when I cultured the HK-2 cells, and I suggest you replace the chamber to have a try. May it helps.
http://www.merckmillipore.com/CN/zh/product/Millicell-EZ-SLIDE-8-well-glass%2C-sterile,MM_NF-PEZGS0816
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