Immunocytochemistry on cells that grow in suspention - is it possible? (Apr/02/2006 )

Hi
I usully do immunocytochemistry (IC) on cells that are grow adherent to the culture vesel but is it possible to do it in cells like lymphoblasts that grow in suspention?
Thanks

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yes. When washing the cell, centrifuge it and remove the supernatant.

I hope this may help.

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and to put them on the glass plate after labeling, use polylysine plates. Put a drop (200 uL) of cells on the plate, let the cells sediment. They will stick on the plate, and then you can wash the plate, and fix the cells, and wash again.

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hi,

better to put some viscous solution (Moviol, Fluo Mounting Med...) after IC

Seb

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Just like that?
It sound so easy! I thought I had to centrifuge the cells in a manner that I think is called citospin, where the glas plate is also used to centrifuge and where the cells stick to the plate during centrifugation.

How can I prepare polylysine plates?
A drop of cells? do you recommend any particular cell density?

Thanks

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In continuance with miselle's suggestion, it is a pretty standard practice to adhere cells on glass slides using poly lysine solutions. These are commercially availble and I think it should be pretty easy for you to get hold of a protocol for coating the slides with poly lysine solution. One easy source of such protocols might be some immunology labs working on T cells/ B cells etc. With regards to cell density, I think you would have to see how crowded your slide looks. You just spread the cell suspension with a pipette tip. Give it sometime to stick. Then rinse off and go in with your staining protocol. It is best tostart with a higher density than what you might need because depending on your staining procedures, you will tend to lose some cells midway. But if you are carefull you will not lose too many.

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Or just resuspend your LCLs at 2,000,000 per ml and pipette a drop of the suspension onto a multi dot slide and then pipette it up again leaving a film of cell. Air dry this in the hood then fix in 2% paraformaldehyde and permeabilise with acetone/ethanol or whatever your standard method is.

Ceri

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