Re: HELP - PLEASE!!!!
|From:||Greg Dobbin <dobbin@Upei.CA>|
You sound rather desperate, so maybe my opinion couldn't hurt. If
your cell suspension is in medium with added protein (like FBS in
cell culture medium), then I would remove that from the media first,
(but remember, because I would do it it doesn't necessarily make it
right! Maybe others have a different idea). This would involve
washing the cells free of the culture media (ie pellet the cells,
remove supernate, replace with PBS or plain culture media, and
repeat). Then resuspend the cells in a similar non-proteinaceous
media to the desired concentration.
The next problem can be the salt crystals that form, when the cell
suspension is left to dry. This is (in my experience) impossible to
remove completely, but the number crystals and extent of related
cell damage can be minimized. Place a drop of cell suspension on
the slide and leave it for 5 mins (less, if drying is noticeable earlier),
and then use suction (I prefer to use a glass pasteur pipet hooked
up to a lab vacuum system) to carefully remove the excess fluid
(place the pipet at the edge of the drop). Most of the cells would
have already settled to the glass and "stuck" so that you are
essentially just removing supernate. Then allow the smears to
completely dry, and then fix.
Having said all that, if you have (or have access to) a cytospin
machine, it makes the best cell smears I've seen. Good luck.
Date sent: Wed, 22 Nov 2000 15:37:05 +1100
From: Bruce Abaloz <firstname.lastname@example.org>
Subject: HELP - PLEASE!!!!
Forwarded to: DOBBIN@acad1.cs.upei.ca
> Hi Dear Histonetters.
> I always couldn't make a good quality smear slide,could you give me some
> idea how to make the smear slide or get a good method to do that from your
> net system.
> What I have done to make the slide is the following:
> the concentration of the final cell suspension is about 3-6 x 10(6)/ml, 5ul of the cell suspension was dropped on the slide and smeared gently to a small circular area, then air dry and fixed in acetone for 10 min, finally,the fixed slide was stored in -80 degree freezer.
> But the quality of the slide is not satisfactory for immunocytochemistry.
> Thanks a lot
> Zhen zhang
> Zoology Dept.
> University of Melbourne
> Parkvelle, 3052
> Tel 61-3-8344 4334
> Fax 61-3-8344 7909
> BRUCE ABALOZ
> DEPARTMENT of ZOOLOGY * PH: +61 3 83446282
> The UNIVERSITY of MELBOURNE * FAX: +61 3 83447909
> Parkville, Victoria.3010 * EMAIL: email@example.com
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