Summary of replies, 96well labelling

Dr. Robert Ashcroft cytomat at netcore.com.au
Tue Dec 22 21:04:08 EST 1998


Fellow travellers,...
At Michael Herron's suggestion (thanks Michael), I am posting the response 
I have so far.
This will satisfy the numerous requests I have had for the summary, as 
well.

Any additional updates I get will be added to the file which will be loaded 
as a html file at the web-site of FuCell, the Sydney company which started 
me off on this.
Louise will likely have that done in the first few days of the New Year. 
(OK Lou?)
The web-site is http://fucell/web.unsw.edu.au.
Best regards and thanks to all the respondents for this little 
treasure-trove.
Bob
Summary of replies received on 96 well plate labelling of cells
Jane Allan
We label in plates.  The only difference in our system is that we don't
aspirate - just invert the plates sharply!  The cells do remain behind. 
 The
minimum number we do for flow  cytometry is 10,000 per well.  We spin in a
96 well plate carrier - Beckman, Hearaus and I think Sigma have these
attachments.
Merry Christmas,
David Leavesley
In spite of your no more request, I'm chipping in with another tip.  If 
after spinning, etc, you still have problems, try coating your 96-well 
plate with poly-L-lysine. Cells stick to it like glue so its fine for 
expression data, but don't expect to get any functional data.  A similar 
trick for proteins is to coat with methylated BSA. The methyl groups react 
with most side-chains in aqueous solution covalently linking your protein 
to the plastic. It also works for cells.
Ciao for niao
Larry Arnold
We routinely do all our staining of cells in round bottom polypropylene
plates from Costar.  Yes there are adapters for centrifuges to hold plates.
Centrifuge and then on upside down flick will throw out the S/N and cells
will stay in plate.  Let me know if you have any ??

Happy Holidays!
Liljana Stevceva
There are two things that you can do: You should order a fitting for
plates for the centrifuge. I usually centrifuge them at 1400 rpm for 10
minutes; before your initial staining, resuspend the cells in fetal calf
serum. Merry Christmas and a Happy New Year!
Best regards,
Joseph Martinez
While we don't use cells, we routinely stain and wash 0.5um polystyrene 
beads in 96 well plates for our flow work.  We use an IEC centrifuge with 
adaptors to hold the plates while centrifuging (in the Fisher catalog).  We 
then use an old ELISA plate washer (hand-held) to wash the beads and 
aspirate the supernatants.  The ELISA plate washer is vacuum and gravity 
operated, made by Skatron in Norway.
Hope this gives you some useful ideas.
Regards,
Heidi Engelhardt
I used to use eppendorf tubes, but once my experiments got too huge, my 
thumbs couldn't take the strain (opening and closing of tubes between each 
step), so I now do everything in 96 well plates.  All my volumes are the 
same as with epps (eg. antibodies added in 50 ul) but the washes between 
primary and secondary antibody steps are a bit reduced in volume (200 ul). 
 The key points:
* you need to buy the adaptors for the plates to fit whatever centrifuge 
you have
* this was the painfully expensive part (we have a Jouan)
* maybe someone in your building/group already has them?
* after each spin, we remove supernatant with a quick "flick" over the sink 
(entered as "spin-flicks" in the protocols)
* a wimpy flick won't remove the supernatant, we have never had     trouble 
losing the cells so be brave
* for the transfer into flow tubes, we add 100-200 ul PBS (or whatever you 
usually use), then let them swish gently for about five minutes on an 
orbital shaker
* this proved to be very important.  without this step, recovery of     the 
cells was poor
* when we were first setting this up, we checked on an inverted 
     microscope to make sure we had them all
* of course, when you pipet them out, draw up and down a bit as    usual

Good luck and merry Chistmas!
Warren Shlomchik
We routinely stain in 96 well plates.  Centrifuges from most manufacturers 
come with 96 well plate adapters.  We stain in inexpensive non-tissue 
culture round bottom plates, wash in 200 microliters, spin for 2-3 minutes 
between manipulations, and aspirate using pasteur pipettes connected to 
wall suction.  We transfer cells using a multichannel pipette to 1.2 ml 
tubes in 96 tube racks (COSTAR).  You can drop these tubes into a standard 
FACs Falcon tube for analysis.
Good luck.
Bruno Gran
staining in a 96-well plate works fine. You can "seed" your cells, wash 2x
in staining buffer, stain with a very low amount of Abs-fluorochrome (you
can do your own titration but we normally use 2.5 to 4 ml of Ab), incubate,
wash 2x, transfer the samples into tubes and acquire. The major centrifuge
brands have appropriate 96-well plate "dishes" (e.g. Sorvall etc.). When
you have washed you can either dump the supernatant or remove it with a
multichannel pipette (in my hands dumping vigorously works better).

Good luck, you need details let me know
Debbie Shapira
We have a new centrifuge with microplate carriers on. it's a Beckman but
all of them have this feature. You might need a different rotor though. If
you want you can come over and try.
Dale Godfrey
I do this pretty regularly and it works for as low as 10000 cells per well, 
although 10^5 or better still 10^6 is recommended.
Use a U bottom 96 well plate, which can be centrifuged using a plate 
adaptor (available with most bench top c'fuges).
Add cells in 100 microlitres or so, spin at about 300g for 3 minutes. 
Remove supernatant by one sharp flick down the sink, leaving the pellet 
intact (dont flick again, or you will lose cells). Vortex the plate 
(resuspends cells), then add antibodies in about 25-30 microlitres. Gently 
swirl the plate with a vortex set on low (dont splash, as this will cause 
crossover between wells). It is important that the cells and antibodies are 
thoroughly mixed of course. I hold the plate firmly on the vortex such that 
the cells in the 30 uL swirl around but do not splash. Wash antibodies out 
with 2 x 200 uL of PBS, spinning down with each wash. You should not lose 
many cells at all with this technique.
David Brown
We have found in our lab that lipids such as Lipofectamine with polyvalent 
cation heads tend to hold down cells quite stridently to the point of 
requiring trypsin/EDTA to get them off. We have used this in the context of 
immunoflurescent staining. Other options are poly-l-lysine or ather tissue 
matrix protein such as collagen.
Hope this is of some assistance
Andrew Collins
Hey Bob!  It sounds like you should be using the LSC.  Its great for small 
numbers of cells, and if you believe Clatch, you can do all your washing 
and staining on the slides.  Very low volumes.  Alternatively, we and lots 
of people have a 96 well plate rack for our centrifuge.
Nick Pearce
I may have miss the point so let me know if I have.  We regularly label 
cells in 96 well plates (normally round but flat will work)for running on 
the Flow cytometer. As you say you need to purchase a bucket (bad word) 
from the maker of your centrifuge that holds 96 well plates. I assume (big 
mistake) that all centrifuge companies make holders that 96 well plates 
fit. I have used Beckman and Hereaus centrifuges to spin 96 well plates. We 
routinely  use 96 well plates as the labelling is quicker than if performed 
in tubes.
Patricia Price
this does work...many centrifuges take plate spinners. We had a convenient
one on a Hetich. Warning you need more wash steps because the volume is
small...so it doesnt always help.

Tricia
Tony Purcell
We use "V-shaped" 96 well plates for staining of large numbers of samples. 
 We don't necessarily push the number of cells per well but routinely use 
5x10e4 to 1x10e5 per well for the staining (the higher number of cells can 
be split into multiple wells for two colour staining etc.).
It's crucial to wash extensively and spin the plates. We have a Beckman GSR 
centrifuge which has micro-plate adaptors. You can spin these up to 1400 
rpm without damaging the cells (at least as determined by exclusion of PI).
Hope the info. is of use.
Cheers
Alan Baxter
Yes. It (the rotor insert for 96 well plates ) will be essential for what 
you want to do. You are welcome to send someone here to see how we do it.
Regards,
Alan
Andy Lidy
Bob ,
In the old days at BD we use to stain in 96 well plate , Call Jin Chen at 
BD and tell her I gave you her name .   Also Shou Shye or Maria Quist can 
help you .  They all work for me when I was at BD.
Andy
PCR TaqMan
PE Applied Biosystems
Carol Wyatt
We label lymphoid cells on a regular basis and immunophenotype them using a 
fairly large number of different mAbs to leukocyte antigens.  To do this, 
we transfer the cells to V-bottom plates, although you needn't do that to 
be successful.  A number of years ago, we obtained centrifuge plate 
carriers that hold 96-well plates.  We use Beckman equipment, but I 
strongly suspect that these carriers are fairly common.  Your best bet is 
probably to call the company that makes the centrifuges you use.
Brian Newsom
Most tabletop centrifuges (Beckman and Sorvall at least) do have adapters 
  for 96 well plates. In the past I have stained cells in them and it 
  usually works well. If you can get the lyse no-wash system to work for 
  what you are doing it would probably save a tremendous amount of cells 
  also.
Martin Dominguez
Simple answer yes you can.
Your current centrifuge suppliers should be able to sell you the right 
bits.
We have used the   IEC Centra with 4 x 96 wellplate carriers for some 9 
years.
http://www.centrifuge-pipette.com/


Simone Cross
Dear Rob,
We use a V-well 96 well plate.  Works really well as you only
need to label for 15mins at 4-8 degrees C (open and close the fridge door a
couple of times = 6-8degrees C)!
And two responses from Phil Stumbles
#1:---
Dear Bob,
We routinely use 96-well plates for FACS labelling without too many 
problems.  You need to use round-bottom plates and you will also need ac  
cess to a refigerated centrifuge that has plate holders - these are 
certainly available for Beckman centrifuges and most other brands as well I 
think. The easiest way to aspirate is via a vacuum tube with a yellow 
pipette tip attached - run the tip down the side of the well and remove 
supernatants without disturbing the pellet.  Loosen the pellet by briefly 
shaking the plate on a plate-shaker and then add 100ul max. of wash fluid, 
shake again and then spin for 2-3 minutes and normal speed.  Unless you 
then have access to a FACSmate 96-well plate sampler, you will then have to 
transfer cells to FACS tubes - not too laborious if you use a multichannel 
pipette.
Hope this helps!
Phil Stumbles.
#2:---
I have to admit I have always been a little nervous about the "flick" 
method -  I just can't bring myself to do it!!  The "babies" are generally 
too precious!
Merry Christmas
Artur Plett
Yes, well, we have a BECKMAN table-top centrifuge and it does have adaptors 
for 96 well plates. (I do most of my staining in 96 well plates and it 
works great. Saves a lot of time when you have 10 samples or more)
Maria Daly

Its possible to stain cells in round-bottomed 96well plates. Then you top 
the wells up with wash buffer, centrifuge plate for 5 mins to pellet the 
cells ( you can buy special adaptors to take plates from the centrifuge 
company). Flick the plates to remove the supernatant ( this requires faith 
that the cells will remain in the pellet! Do it sharply!) Shake plate on a 
plate shaker ,briefly, to resuspend pellet and repeat wash step.
Good luck







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