multiwell autosampler - washing

Giovanna Borsellino gborsel at tin.it
Thu Dec 13 10:18:51 EST 2001


We do the same - except after spinning the cells we simply flip the plate
over the sink with one vigorous move - fluid comes out and cell pellet
stays were it is! Fast and guaranteed. Must use V-bottom plates.

Giovanna


At 08.59 12/12/2001 -0500, Laird Bloom wrote:

>         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
> >
> >

Giovanna Borsellino, MD PhD
Neuroimmunology
IRCCS Santa Lucia
Via Ardeatina 354
00179 Rome
Italy

Tel. ++39.06.51501521 (office)
       ++.39.06.51501552 (lab)
Fax ++39.06.51501553

e-mail: gborsel at tin.it




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