University of Pittsburgh Cancer Institute (UPCI)

CCSG Acknowledgement

Required CCSG Acknowledgement

The NCI requires that publications acknowledge the UPCI CCSG support, and they are tracking compliance. If a UPCI CCSG-supported Shared Resource provided data used in your publication, please include the following statement in the acknowledgment section of your publication(s):

"This project used the UPCI [insert name(s) of shared resource(s)] that [is/are] supported in part by award P30CA047904."

Shared Resource Directors: Please make sure to include this statement on all of your order forms, contracts, etc. as a reminder to your users to acknowledge the UPCI CCSG support.


FAQs

HOW LONG WILL IT TAKE ME TO SORT?

Calculation for the 70 micron nozzle (lymphs, PBMC, bone marrow, yeast):
Nominal maximum throughput (70µm) = 20-25,000 events/second ~72 - 90(10)6 events/hour
Net throughput (corrected for normal recovery/yield estimates) = 70% ~50–63(10)6 events/hour
Estimated throughput = 50(10)6cells/hour (50 million cells per hour that can be run through the machine)
For most cell lines, a 100 micron nozzle is needed, which reduces throughput by at least 70% – adjust calculation accordingly.

Collection rate, 70µm (maximum) = (viability) x (expression) x 50(10)6 events/hour

EXAMPLE:
Attempting to sort out a sub-population (5%) from your cell line.
The number of cells you can provide is unlimited, and the viability is 90%.
A pure, sorted population of at least 5(10)6 cells needs to be recovered. Given the estimated sorting capabilities (see the calculation above), estimate sorting time as follows:

Collection rate (maximum) = 0.9 (viability 90%) x 0.05 (expression 5%) x 50(10)6 events/hour = (0.9) x (0.05) x 50 million cells/hour = (.045) x 50(10)6 = 2.25(10)6 = 2,250,000 per hour.
100 micron nozzle reduces by at least 70% - adjust calculation accordingly.

Recovery of 5(10)6 total =[5(10)6 ÷ 2.25(10)6] = 2.22 hours of sorting, at minimum. Add an additional 45 minutes to sorting time to set up the machine.
However, please assume that something might go wrong, and you will need twice the number of cells, and it will take twice as long.

REMEMBER: 4 SEPARATE POPULATIONS CAN BE COLLECTED AT THE SAME TIME.

FOR ANY SORT, YOU WILL NEED TO BRING THE FOLLOWING:

  1. Control(s):
    • Negative control
    • Compensation controls if needed
    (If you have any questions about appropriate controls, please ask your friendly flow cytometry technician.)
  2. Sort sample(s):
    Bring controls and samples in 12 x 75 mm sterile; snap-cap; POLYPROPYLENE tubes, with no more than 5% serum.
    The control(s) only needs to be 300 uL final volume, with at least 300,000 cells. Sample should be re-suspended at a concentration on 30-40(10)6 cells/ml.
  3. Collection tubes (4 separate populations can be collected at the same time):
    For each tube for collecting sorted cells, fill the tube ¼ full with 50% serum/media. You may use either 12x75mm tubes or 15 ml tubes.
    (12x75mm tubes are needed for 4-way sorting - fill the tube ¼ full with 50% serum/media.)

    Cells to be collected exit the machine at a concentration of ~1,000,000 cells/ml using the 70 micron nozzle; 500,000 cell/ml using the 100 micron nozzle. You can now calculate how many collection tubes you will need for each population collected. (Example: For 70µm nozzle, you place 4ml media/serum in the bottom of a 15ml tube, leaving 11 ml empty volume for collecting cells = 11ml x 1,000,000 cells/ml = 11(10)6that may be collected in that tube.)

FOLLOWING THE SORT
For maximum recovery, fill collection tubes to the top and let sit 30 minutes before spinning them down.