[Cytometry] sorting into Terasaki plates

Barsky, Lora lbarsky at usc.edu
Thu Jan 21 14:26:39 EST 2010


Hello Lynda,

I do not have experience sorting into a 60well Terasaki plate nor a 384well plate, however, I have built custom templates for the Vantage DiVa when a 96 well plate did not track well with the programming that the software provided.

It would seem to me that if the 60 well plate did not fit securely onto the plate support provided by BD, you should have your machine shop build a platform (to be placed between your 60well plate and the existing plate support) that you could securely mount the 60well plate to.   The outer dimensions of the platform will have to be that of a standard 96well plate so that it locks into the standard plate support.   Of course, be mindful of of your vertical clearance.  If I remember correctly, there isn't much vertical clearance to begin with after the plate shield is installed.

The custom plate programming allows you to define position A1 and then the last position, lets say F12, if that is the case.  You should also see fields to enter the number of columns and rows.   Then you have to program the center of A1 by setting home and also you have to program the center of the last row and column's well.   Then the software divides the coordinates in the x and y direction by the number of wells you have defined.    Obviously, test, test and test again, before running the sort.   

What worked well for me, was to program the sort layout to sort 50 events to every well on the plate and sort onto a plate with the lid.   The lid was key, so that I could see the dried droplets and I could determine if I was close enough to center for the end user to feel good.   (Sorting onto the lid,  doesn't account for the final placement completely, as the cells are coming down at an angle and there was a little bit more of a vertical drop until the plate bottom was reached.   for your new system of using either a 60well or an 384 well plate,  you may want to fire up the fluorescent scope and sort directly into the wells, then check each well for the 50 sorted beads and determine for yourself that you can hit the middle of the wells.

On another note,  I did have the pleasure of working with a very skilled technician who could do single cell PCR from sorts run on our Vantage DiVa and we used the ACDU programming to sort 1 cell directly into eppendorf tubes for that case.   Another highly skilled technician was able to single cell cloning of adult stem cells from UCB and BM, for that we used 96well plates filled with 100um volume complete with feeding cells that were plated prior to our sorts.

Sorry I haven't the time to address your other questions.  Good luck.
Lora

Lora W. Barsky
Manager, Flow Cytometry Core
Eli and Edythe Broad Center for Stem Cell and Regenerative Medicine
1450 Biggy St, HNRT4513
MC 9601-NRTLG591
Los Angeles, CA  90033
phone (323) 442-7942
On Jan 21, 2010, at 7:01 AM, Guzik, Lynda wrote:

> Hello All,
> 
> I have a FACSVantage Diva.  I have been asked to sort into Terasaki plates (Nunc brand cat#439225) - you know - those very tiny 60well plates that hold about 25ul/well.  This would be for a clonal sort - so one cell/well.  I know that I will have to define the plate in the software.  Does anyone have any experience sorting into these plates?  If so, can you offer any advice regarding the following:
> 
> 1.  ensuring consistent plate alignment so that the sort stream is correctly targeted (multiple plates will be sorted)
> 2.  making sure the plate doesn't shift as the platform moves during sorting
> 3.  the appropriate well volume for collection (many years ago, I used these plates for proliferation assays at 20ul/well, incubated in a hanging drop formation in individual humidity chambers - the wells were surprisingly stable, but I don't know about sorting into that volume...)
> 4.  appropriate collection media to optimize cell viability
> 5.  maintaining stability of the volume in the wells during the sort and during transport to the investigators lab...
> 6.  and any thing else that I may not have thought of yet?
> 
> I have suggested using 96well V-bottom plates but the investigator feels he won't be able to locate the cell in the well, even after spinning the plate.  Would a 384well plate be a better choice?  Are there any specific caveats with the 384well plates?  Any and all advice will be greatly appreciated!
> .
> Thank you!
> Lynda Guzik
> McGowan Institute - Lagasse Labs
> Rm 520A - Bridgeside Point Bldg
> 100 Technology Dr - Suite 200
> Pittsburgh, PA 15219
> 412-624-4456
> guzilj at upmc.edu<mailto:lguzilj at upmc.edu>
> 
> 
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Lora W. Barsky
Manager, Flow Cytometry Core
Eli and Edythe Broad Center for Stem Cell and Regenerative Medicine
1450 Biggy St, HNRT4513
MC 9601-NRTLG591
Los Angeles, CA  90033
phone (323) 442-7942





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