Hi Gayathri,

Follow the protocol attached.

Hope that helps,

Best-

Hello Sir,

Thank you so much for your link.

@ Amritlal Mandal

Hello Sir,

I have prepared the gelatin coated cover slips according to the protocol and allowed them to dry and then I have placed them in the 6 well plate on the same day (everything was done under aseptic conditions and I skipped the addition of water step as I needed them on the same day). When I added the media, i found that little gelatin dissolved into the media. After two days i got severe fungal contamination and Control well ( with media and HT-29 cells) is free of contamination. Is this because of dissolved gelatin? How to over come this problem??

Hi Koula,

I feel your pain... Been there, done that...

So, getting adherent cells to grow/survive/culture on glass coverslips is easy.... IF you know how.  And if you know how, it's probably only because you've spent many tedious and painstaking hours figuring it out...

I have done this with different cells and here's what I've found.  The exact method needed, varies with the type of cells you are culturing, but the idea is the same.

You need to coat this glass with something that the cells will adhere to.  You want to avoid infecting the system, AND you want to be able to use the sample for microsocopy.

The reagents I'm familiar with are:

1: Fibronectin

2: Laminin

3: Collagen

4: Poly-d-Lysine

5: Gelatin

What I have seen is that different reagents work for different cells.  You have to simply try them.

Now, there are coverslips that are pre-coated.  you might consider just purchasing them and save yourself the heartache.  Fisher Scientific and Corning have these.

As fate would have it, I am about to try to culture Prostate cancer cells on glass cover slips.  I am considering using a combination of fibronectin and poly-d-lysine, OR laminin and poly-d-lysine...

i hope this information helps you...

Good luck!

Hello Sir....

Thanks for your kind replay...I agree with you. It differs from cell line to cell line. I have standardized the protocol with slight modifications by using gelatin and it worked well. I have completed that experiment with positive results.

Once again Thank you for your time.

Hi Koula,

I am also having issues growing HT-29 cells on glass. Would you mind sharing your protocol? I have to do several washes in my procedure, which unfortunately are necessary, and the HT-29 cells wash off the glass slide.

Any help is appreciated.

Thank you.

@ Kristie,

Hi, I can understand your problem. Here is a modified protocol which helped me a lot.

Weigh one gram of gelatin and add to 5-10 ml of water and heat it until gelatin dissolves and it forms a clear viscous solution. Allow the gelatin solution to become lukewarm.

Place the coverslips in a petriplate (100mm) and add 100 micro liters of luke warm gelatin solution on each coverslip and spread it uniformly. Carry out this step in laminar hood. 

Allow the coated coverslips to dry overnight. You can store them in 4 degrees for further use.

For adhesion, Place a gelatin coated cover slip in each well (6 well plate) and put on the UV light (Laminar Hood) for 20 minutes. 

Wash each well, containg coated cover slip with 1X antibiotic solution once and with PBS twice.

Now seed the wells with HT29 cells.

 While adding and aspirating the used media or washing, do it gently, from the  corners of the well. This will minimized washing off cell lines.

or you can either use a 60 mm dish instead of a 6 well plate or transfer your cover slip containg HT29 cells into a 60mm dish for washing. This will definitely mimimise washing off of cell lines.

Good luck.

Thank you so much!!! 

Hi, Koula! can i use poly-l-lysine instead of gelatin in similar described way?

Tulsi Joishy, I would use poly-d-lysine. Poly-l-lysine can be taken up by the cells. I use cell culture grade poly-d-lysine. I coat with 200 uL per chamber in a 4 chamber slide (the well surface area is slightly smaller than that of a 24-well plate) for 30 mins to 1 hr. Rinse with PBS, not water, and you can seed immediately. If you don't have cell culture grade and it's packaged similarly to the gelatin, then I would follow Koula's steps for sterilization.

Just suggesting-

You could try chambered cell culture slides

Thank you Kristie. I will follow suggested methods but how much conc. do you use.