Suzanne T. Ildstad, M.D.
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MATERIALS

1. Autoclaved Millipore H2O
2. Biosure Flow Cytometry Sheath Solution 8X (Cat# 1020)
3. flow cytometer/ sorter (EX. FACSVantage SE)

SORTING

1. Align sorter using an alignment standard (EX. CRBC)
2. Check drop delay setting with fluorescent beads, which can be counted using a fluorescent microscope (EX Polysciences Cat#18141).
3. Clean sample line by running 10% bleach for 10 minutes (avoid EtOH).
4. Run Cell Sort Media (from staining protocol) for 5 minutes to flush any bleach left in line.
5. Set compensation using single color B220 FITC and B220 PE bone marrow stains to provide a positive and negative population for each of the fluorochromes used in the FC stain. This stain also requires setting the lymphoid light scatter gate (FIGURE A).


B220 FITC B220 PE
FIGURE A

Note: Top cytogram shows Lymphoid gate. Top green shows correct FITC compensation and top orange correct PE compensation using B220 single stained bone marrow. Second cytogram in each column shows overcompensation, while third shows undercompensation. Correct compensation is very important for correct analysis of FC stain.

6. After compensations are set, filtered FC are ready to be sorted. Again plot FSC vs SSC and set a wide Lymphoid gate (see Region1 FIGURE B).


FIGURE B
7. Now create a Region 1 gated cytogram with TCR FITC (x axis) vs CD8a PE (y axis). A staining pattern similar to that shown in FIGURE C should be seen. Set a large gate around the CD8a positive/ TCR negative population (See Region 2). CD8a positive/ TCR positive cells (T CELLS) could be sorted at the same time (See Region 3). The FC gate must satisfy both Region 1 (light scatter Figure B) and Region 2. The T CELL gate would satisfy both Region 1 and Region 3.
8. The FC cells should be sorted into 12x75 mm tubes containing 1 ml of CSM or other osmotically controlled media. Sera seems to help maintain viability. Our lab typically collects 30,000 FC per collection tube (enough for 1 transplant).
9. Our lab generally sorts FC at 2500 to 3000 cells per second. Also, sorted cells and presort tubes are kept on ice while not on the sorter.


FIGURE C
10. After the sort is finished each sorted tube is reanalyzed for purity. It is a good idea to run CSM alone for 10 minutes after sort to prevent presort cells left in the line from contaminating the reanalysis. We analyze 200 cells per reanalysis. FC sorts generally yield 90-95% purity while the T Cell sorts give better purity (95-99%). Figure D shows examples of resorts for both populations. Expect some dead cells (low forward scatter) in the FC sort, while the T cell sorts have less dead cells. In general we do not try to recount sorted cells as the instrument counts on the Vantage are fairly accurate.

Science from Bench to Bedside