http://www.protocol-online.org/forums/page/index.html/_/cell-culture/ Fri, 10 Feb 2012 08:52:45 +0000 43200 http://www.protocol-online.org/forums/page/index.html/_/cell-culture/how-to-coat-coverslips-before-seeding-cells-r21 jennycharlies Posted 18 August 2011 - 06:55 AM
Hi,

I am going to seed my cells onto coverslips inside the wells of a 12 well plate and was wondering what is the best way to coat the coverslips?

I have 18mm coverslips and need to coat with poly-D-lysine. But presumably I need to sterilise the coverslips first.. What is the best way to go about this? I assume I need to coat the slides while in the wells of the plate?

Thanks for your advice!!

Chakchel Posted 18 August 2011 - 11:10 PM

Hi!

Yes, you first should sterilize your coverslips. You can do this either by putting them into the autoclave, e.g. wrap several in a little "bag" of aluminium foil, or by dipping them in Ethanol and shortly flame them (too long flaming will let them burst).

Then you can directly cover them in your well. Put your poly-D-lysine in the well, so that the coverslip is covered, but not too much, then let it dry in the hood for several hours. If then there is still some liquid, you can suck it of.
Finally wash the wells once or twice with PBS.

Done, and ready to use.

Edited by Chakchel, 18 August 2011 - 11:11 PM.

fysio lab Posted 19 August 2011 - 02:25 AM
Hello,

we put non-sterile coverslips in a glass petridish, wrap that in alu-foil, autoclave it and before use we put the CS in the wells with sterile forceps. After that we put our coating solution on the CS (in our case 2% gelatin), we leave it 30' in the incubator. Then we remove the gelatin and let it dry for a few minutes in the flow, lid open... meanwhile we split and count our cells. All together this doesn't take much time.
Good luck]]> Mon, 22 Aug 2011 20:24:36 +0000 4c56ff4ce4aaf9573aa5dff913df997a http://www.protocol-online.org/forums/page/index.html/_/cell-culture/tips-on-freezing-and-thawing-of-cells-r18
I am facing problems in thawing of cells. The procedure which we follow is- we take stock from liq. N2 then though it in water bath at 37 degree. Then we take medium in falcon (5ml) then add thawed cells dropwise into it. We then centrifuge it at 1500rpm for 5 mins.
but the problem is that after thawing i am unable to get viable cells.
Almost all cells are dead. So what all modification shall i make ? Its really hard to revive cell line from frozen stock?

The general rules for freezing and thawing cells is: freeze cells slowly but thaw cells quickly. It seems to me that you have not frozen your cells in a gradual manner. There are many ways for achieving gradual decrease in temperature. Check the section for cell preservation on this site and you will find protocols of different flavors for this purpose.
http://www.protocol-...tion/index.html

One thing you could possibly try is not to spin your cells straight away, just add the warm medium, and let them sit over a few hours to overnight, then wash out DMSOcontaining medium, and feed with normal medium plus serum (increase serum conc to 10 or even 20% for a little while). Sometimes brittle cells get sheared while centrifuging, so that's why the modified protocol. DMSO is toxic though, so change medium as soon as you see under the microscope that your cells have settled.
If that's the problem you could instead decrease your spin time- even 1min at 1500 should pellet enough cells.
Also, I add medium to my cells, no vice versa, I'm not sure if that would make any difference at all.
As for freezing cells- I do it your way, it seems fine, but your cells could be very sensitive, so could be the problem.

Though my experience of handling the cells is less than 2years. I would like to suggest you some things. Even though if you leave DMSO in the media after thawing the cells the dmso in low cocentration is not toxic 2 cells more over once you dilute the small volume of cell suspension in 5-10 ml of media, it further dilutes the dmso. It looks to me like the protocol you are following may be causing the cells to die, I think you recognise the fact that centrifuging at high speed can lead to cell necrosis. One method is to lower the speed of centrifuge maybe 900-1000 rpm for 4-5 min should be adiquate.
Alternatively you should also consider the method you are using for preserving the cells. If you freeze the cells directly in liquid nitrogen this can also lead to cold shock induced cell lysis or damage the membrane, one way of preventing this is gradually lowering the temperature such as storing 4 degrees for few hours then transfering to -20 degree for overnight and then storing in -70 degrees for an couple of days then transfering to liquid nitrogen tanks, by following this method u r not only making the cells to adjust to the lower temp you are also ensuring that they donot undergo cold induced necrosis leading to cell membrane damage and lysis.


This is how to freeze and thaw cells:

Always check the cell viability before freezing. They should be highly viable: about 95%. keep your freezing media ( 10% DMSO in FBS) cold. Centrifuge cells for 1000 RMP for 5 minutes. Prepare labeled cryogenic vials. Cell concentration should be about for e.g. collected from a 50 ml flask, about 50 million cells. You can have 10 vials and about 0.5 milion cells per vial. After you centrifuge, get rid of media completely, gently tap the pellet to make it loose. Add 5 mls of cold freezing media resuspend with a pipet and transfer 0.5ml to each vial. Close the cap tight and place cryogenic tubes in special cryogenic container that has alcohol at the bottom and cool down gradually, they usually hold up to 20 vials. Close the top and transfer the container to -70 freezer. Wait 24 hours and no longer than 15 days before transferring them out of the container into the liquid nitrogen boxes.

To thaw the cells, be very quick, take the viall out still with some liquid nitrogen, walk to the water bath. Take the vial, make sure the cap is very tight, sometimes it becomes loose. thaw the vial holding the opening upward so the water from the water bath does not contaminate cells or if there is some detegent in the water it doesn't become in contact with the inside of the vial . When frozen cell media is almost half thawed (about few minutes), take it under the hood and add 0.5 warm media to the cells and immediately transfer to a flask with about 10 to 15ml warm media in it. DMSO will be diluted and you can check the viability.]]> Sun, 15 Aug 2010 01:50:14 +0000 5ef059938ba799aaa845e1c2e8a762bd