P. Keller 2007

Part 1: Make single cell suspension of freshly dissociated cells

  1. Thaw 1 vial 618 CP and plate in RMFC for 1-2 hours
  2. Remove non-adherent + 1-2 PBS washes to 50 ml conical, spin down cells
  3. Resuspend pellet in 10 ml cold PBS, pass through 18G needle 8X, spin
  4. Resuspend cells in 2 ml T-EDTA, pipet up and down for 1-2 min, incubate at 37C for 10’, pipet up and down with p1000 to break up clumps
  5. Add 5 ml B3 + 0.1 mg/ml DNase I (add 140 l of 5 mg/ml stock), incubate 5-10’ at 37C
  6. Add 8 ml of cold RMFC, filter though a 40 m mesh, wash with 10 ml of RMFC, spin down cells, reuspend in 5 ml MC
  7. Remove 10 l and dilute in 10 l trypan blue and count (multiply number by 2)
  8. Set up four tubes A: 50K/gland (150K cells) B: 10K/gland (30K cells) C: 5K/gland (15K cells) and D: 1K/gland (3000 cells), keep on ice

Part 2: Make a single cell suspension of cells grown in culture for 7 days

  1. Wash 1 plate of 618 RMEC with PBS and add 1 ml Trypsin, incubate for 2-3 minutes at 37C
  2. Collect cells and inactivate Trypsin with 10 ml RMFC, spin down
  3. Resuspend cells in 5 ml MC
  4. Remove 10 µl and dilute in 10 µl of trypan blue and count (multiply number by 2)
  5. Set up four tubes E: 50K/gland (150K cells) F: 10K/gland (30K cells) G: 5K/gland (15K cells) and H: 1K/gland (3000 cells), keep on ice

Part 3: Prepare mammospheres

  1. Collect media with spheres plus one PBS wash from 2 plates (7-day-old spheres), spin at 800 rpm to pellet
  2. Resuspend spheres in 1 ml, remove a 10 µl aliquot and count spheres per 10 µl, multiply by 200 to get total spheres
  3. Set up four tubes I: 200 spheres/gland (600 spheres) J: 100 spheres/gland (300 spheres) K: 50 spheres/gland (150 spheres) L: 10 spheres/gland (30 spheres), keep on ice

Part 4: Prepare RMFs

  1. Wash plates of primary RMFs with PBS and add Trypsin for 2-3 min
  2. Pool all RMFs together (need 9 million total), note: from patient ID 615, 612 and 618 with time in culture varying from 7 to 15 days all under 2 passages, spin down to pellet and resuspend in 12 ml RMFC
  3. Count 10 µl of cells and multiply by 12 to get total
  4. Aliquot 750K cells per tube, spin down cells with each tube of MECS/Spheres and transfer pellet to an epi tube
  5. Spin down cells at 1000 rpm in epi tubes, aspirate off all liquid

Part 5: Prepare Col:Mat mixture/Inject

  1. Dilute 10.59 mg/ml collagen (rat tail) to 4 mg/ml with 0.02 N GAA (0.5 ml collagen + 1.32 ml 0.02 N GAA)
  2. Check pH, neutralize the mixture with 0.1 N NaOH until pH is near 6 or so
  3. Make up 3:1 collagen Matrigel by adding 500 l MG to 1.5 ml neutralized collagen, check pH
  4. Add more 0.1 N NaOH as needed to adjust pH to 7-7.5 (pale pinkish color)
  5. Keep on ice and re-check pH before using to resuspend cells
  6. Resuspend cell pellets in 95-100 l of mixture, keep on ice for injection
  7. Inject 35 l per gland into 12 mice

Mouse number /

Cells injected

/ Notes
A: 50K/gland fresh
B: 10K/gland fresh
C: 5K/gland fresh
D: 1K/gland fresh
E: 50K/gland 7 day
F: 10K/gland 7 day
G: 5K/gland 7 day
H: 1K/gland 7 day
I: 200 Spheres/gland
J: 100 Spheres/gland
K: 50 Spheres/gland
L: 10 Spheres/gland

Part6: Cytospin remaining cells for IF

  1. Prepare cells and spheres so at approximately 50K/100 l (500K/ml) and 50-100 spheres per 100 µl (500-1000/ml)
  2. Spin 100 l per spot, let air dry
  3. Fix in ice cold methanol for 10’ at –20
  4. Let cells air dry and place at –80
  5. If still leftover cells, pellet, aspirate off supernatant and snap freeze for RNA at a later date