multiwell autosampler - washing

Laird Bloom LBloom at Phylos.com
Wed Dec 12 08:59:49 EST 2001


	We work in V-bottom 96-well plates and wash by bringing the volume
up to about 150 microliters per well, then spinning the plates at 1500 rpm
for 1 min. in a benchtop centrifuge with plate carriers.   This procedure
gives a nice cell pellet concentrated at the bottom of the well, and it's
easy to remove liquid with a multichannel pipette without disturbing the
cells.	We've also worked with round-bottom wells, but the cell pellet is
more spread out and it's easier to remove cells by mistake.  For most
staining steps, we use 25 microliters of antibody solution and dilute this
with PBS prior to the first spin, then do two washing steps with 150
microliters of PBS each.  The force of the liquid coming out of the pipet is
enough to resuspend the cells at each washing step.  After removing the
final wash buffer (but prior to adding the next antibody), we vortex the
plate gently for a few seconds to loosen up the pellet and then add the
antibody solution with a multichannel pipet, cover the plate, and vortex
gently again.  This is enough to resuspend the cells.
	We haven't tried aspirating with a multichannel vacuum system, but
it should work with the V-bottom plate if the vacuum isn't too strong.


Laird Bloom

Phylos, Inc.
128 Spring St.
Lexington, MA  02421
tel. (781) 862-6400 ext. 253
fax (781) 402-8813
www.phylos.com



> ----------
> From:		Reed, Doug S Dr USAMRIID
> Sent:		Monday, December 10, 2001 3:53 PM
> To:	Cytometry Mailing List
> Subject:	multiwell autosampler - washing
>
> We've just gotten in our multiwell autosampler and I'm looking for some
> advice from those who have been using them for a while. I don't (at this
> point) need any help with the system itself, but what I am interested in
> is how people are washing the cells in the plates. Do you do it manually
> or are you washing with an ELISA washer. And how do you get rid of the
> supernatant between washes?
>
> Thanks!
>
> Doug
>
> Douglas S. Reed, Ph.D.
> Principal Investigator
> Respiratory and Mucosal Immunity
> Department of Aerobiology & Product Evaluation
> Division of Toxinology & Aerobiology
> U.S. Army Medical Research Institute of Infectious Diseases
> 1425 Porter St. Ft. Detrick
> Frederick, MD 21702-5011
> 301-619-6728
> 301-619-6911 (Fax)
> doug.reed at det.amedd.army.mil
>
>



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