Suzanne T. Ildstad, M.D.
Intro | Research Interests | Biosketch | Selected Publications | Staff | CV | Protocols |

I. Materials and Reagents:

Vantage Cell Sorter/Turbo Cell Sorter
Pipet-aid
p10, p20, p200, p1000 pipetmen
Refrigerated Centrifuge
Biological Hoods (The Baker Co. Model SG-600
Microscopes (Leica Model # 020-518.000)
Vortex (Genie 2)
Forceps
Scissors
Sterile 100 micron nylon mesh
Petri dishes
15 mL conical tubes (polystyrene)
12 x 75 mm sterile capped flow tubes
Sterile 10-200 mL yellow tips
Sterile 200-1000 mL blue tips
3 mL syringes
18 gauge needles
22 gauge needles
Pipets, 1 mL
Pipets, 5 mL
Pipets, 10 mL
Pipets, 25 mL
HEPES (1 M)
Fetal Bovine Serum
Hanks buffered saline solution (HBSS, 1X)
Medium 199
Gentamicin (10 mg/mL)
0.4% Trypan Blue
hemacytometer
Anti - adTCR - FITC mAb
Anti - gdTCR - FITC mAb
Anti - CD8a - PE mAb
Anti - B220 - FITC mAb
Anti - B220 - PE mAb
Rat IgG2a- FITC isotype control mAb
Rat IgG2a - PE isotype control mAb

II. Solution Preparation:

1. Chimera Media: Prepare the following using sterile technique. Add 2.5 ml Gentamicin (10mg/ml; final concentration in media = 30mg/ml) into a 500 ml bottle of Medium 199.
2. Cell Sort Media: Prepare the following using sterile technique. Add 5 ml of 1 M HEPES (final concentration = 10 mM) to a 500 ml bottle of 1X HBSS. Add 2.5 ml of Gentamicin (10 mg/ml; final concentration = 30 mg/ml) and 10 ml of FBS (heat inactivated) (Final concentration = 2%). Store at 4° C. Expires after 7 days.

III. Procedure:
NOTE: all procedures are conducted using sterile technique

1. Harvest Bone marrow Cells
   1. Remove the femurs and tibias from 5-8 week old donors and place bones in Chimera Media, on ice. The bones should be free of muscle and fatty tissue. Cut the bones just before flushing to eliminate a loss of BMC. Keep bones on ice at all times until processing.

   2. Flush the tibias and femurs with a 22 gauge needle using a 3 cc syringe filled with Chimera Media. (Depending on the number of donors used, try not to use more than 15 ml of Chimera Media when flushing bones so that all of your sample will fit into one 15 ml conical tube.) Resuspend the BMC using the 18 gauge needle and 3 cc syringe by flushing the suspension up and down. Flush forcefully enough to break up clumps, but not so forcefully that you damage cells. Filter the cell suspension through 100 micron nylon mesh into a 15 ml conical tube using the same 18 gauge needle and syringe. Keep the sample and the media on ice at all possible times.

   3. After processing and filtering, spin the BMC suspension at 1000 rpm, brake on, 4°c, for 10 minutes in a refrigerated centrifuge. Note: bone marrow cells are sensitive to speeds greater than 100 rpm. Pour off the supernatant and gently resuspend the cells by ratcheting. Add approximately 1 ml of CSM. Pipet up and down to make sure that the cells are adequately resuspended.

   4. Determine the number of cells harvested and their viability by using a hemacytometer. Take 10ml of cells and stain with 990ml of 0.1% trypan blue. (Dilute 0.4% stock trypan blue to 0.1%, or 1:4 dilution, with D-PBS.) For a clearer field of view, you may lyse the red blood cells by mixing the 10ml aliquot of cells with 10ml ACK lysing solution and incubating for 2 minutes; then add 980ml trypan blue and count. After the count is completed, adjust the concentration to approximately 100 x 106 cells/ml. Note: each donor mouse will yield approximately 30 x 106 BMC.
2. Staining (NOTE: ALL AMOUNTS OF mAbs ADDED ARE BASED ON OUR TITRATIONS ON BONE MARROW. ADJUST APPROPRIATELY.)
   1. For control tubes, add 20ml cells (approximately 1 x 106 cells) to each of the three control tubes outlined below. To each of these control tubes, add 20ml of the appropriate diluted antibody.

      TUBE 1: FC IC (Isotype control)
      TUBE 2: B220 FITC
      TUBE 3: B220 PE

   2. To the main sample (for each 100 x 106 cells), add:

      anti-CD8a-PE: 25ml straight
      anti-ab TCR-FITC: 10ml straight
      anti-gd TCR-FITC: 10ml straight

   3. Incubate all stained tubes for 45 minutes at 4°C in the dark (covered and on ice). Gently mix tubes every 15 minutes during incubation.

   4. Wash twice with cold CSM: add 10 ml to the main sample and 3 ml to each control tube and centrifuge at 1000 rpm at 4°C for 10 minutes. Decant, blot and ratchet to break up the pellet.

   5. Resuspend the cells with CSM to a concentration of 10 x 106 cells/ml. (So, if you have stained 70 x 106 cells, resuspend your sample in 7 ml; if you have 90 x 106 cells, resuspend in 9 ml, etc.)

   6. Filter the resuspended cells once more through sterile 100 micron nylon mesh.

   7. Changing pipets, further dilute the cells by aliquoting 1 ml amounts into sterile snap cap flow tubes containing 3 ml cold CSM. (Each 1 ml aliquot contains 10 x 106 cells. After aliquoting 1 ml into 3 ml of CSM, each sort tube will have a final concentration of 2.5 x 106 cells/ml, or 10 x 106 cells total in the tube.)

   8. Resuspend each control tube in 1 ml CSM.

   9. Prepare unmarked collection tubes by adding 1 ml cold CSM to each tube, returning the snap cap. Vortex or swirl the tubes to "coat" the sides of the tube with media so as to minimize cells sticking to the sides during the sort. Generally, prepare about double the amount of collection tubes as sample tubes.

   10. Take all tubes on ice, with a lid, to the flow cytometry room.
3. Collection of FC by Vantage Flow Cytometers
   1. The sort operator will gate on the lymphoid region, collect the cells, and analyze their purity at the end of the sort.

   2. FC ARE CD8+/TCR-
4. Injection preparation
   1. Centrifuge sorted cells at 1000 rpm at 4°C for 10 minutes. Decant, blot and ratchet.

   2. Using Chimera Media, pool the cell types together (ensuring uniformity in transplants) and then divide into appropriate injections.

Science from Bench to Bedside