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Notes

    • Thaw and/or prepare prior to start of experiment
        • Mouse media + supplements
            • DMEM/F12 (1:1)
            • 5% CS
            • 10 µg/mL insulin
            • 5 ng/mL mEGF
            • 5 µg/mL hydrocortisone
        • Digestion Mixture (10 mL/sample)
            • Mouse Media
            • 5 mg/mL collagenase (100 uL of 150 mg/mL stock)
            • 125 U/mL hyaluronidase (100 uL of 12500 U/mL stock)
        • Dispase Mixture (10 mL/sample)
            • Mouse Media
            • Dispase II (50 mg/sample)
            • Dnase I (200 uL of 5 mg/mL stock)
            • NOTES: Mix media & Dispase II, then filter sterilize (40 uM). Do not add Dnase I until just before use.
        • 25% trypsin-EDTA
        • PBS + 2% CS

Mammary Epithelial Cell Dissociation

    • Gland dissection
        • For dissection prior to:
            • Thaw samples in water bath for 1-3 min
            • Add to 10 mL of digestion mixture
        • For dissection day of:
            • Sacrifice mice and fill out mouse sample info sheet
            • Dissect mammary glands (#3-5), removing lymph node on abdominal gland, and place in PBS on ice
            • Transfer all glands from one mouse to a 10-cm plate and chop briefly with scissors
            • With a cut p1000 tip on a pipette, resuspend glands with 1 mL of Digestion Mixture, transfer to flat-bottomed conical tubes, and add remaining 10 mL of Digestion Mixture
        • Allow samples to digest for 30-90 min (usually 60 is sufficient) at 37°C on a rotator, checking periodically (every 30 min) to prevent over-digestion
        • Pellet digested glands at 1200 rpm for 5 min
        • Transfer fatty layer at top of supernatant to another tube with 10 mL of PBS and shake/invert to dislodge trapped organoids
        • In originally pelleted tube, aspirate and discard supernatant and resuspend organoids in 10 mL PBS
        • Spin original and fatty tubes for 1 min at high speed to settle organoids, then aspirate supernatant
        • Pool organoids together, resuspend in 5 mL of red blood cell lysis buffer, and incubate for 2 min
        • Add an additional 5 mL of PBS and pellet
        • Wash with 10 mL PBS and pellet
        • Resuspend in 1 mL 0.25% trypsin-EDTA and pipette up and down vigorously for ~1 min
        • Incubate at 37°C water bath for 4 min, then repeat pipetting for ~1 min
        • Add 10 mL of Dispase Mixture per sample and mix for ~1 min by pipetting
        • Filter digested cells through a 40 uM mesh filter into 50-mL tube, transfer to 15-mL tube, & pellet at 1200 rpm for 5 min
        • Resuspend cells in 1 mL of PBS + 2% CS and count cells

FACS Staining

    • If not sorting Make aliquots of 400-500,000 cells in FACS tubes for unstained/control samples and 200,000 cells for stained samples, spin down, resuspend each in 200 uL of PBS + 2% CS.
    • If sorting: From sample with largest cell #, make aliquotes of 200k cells for unstained/control samples, spin down, resuspend in 200 uL of PBS + 2% CS. Leave samples that are to be sorted in original volume in 15-mL tubes when applying stains.
    • Add antibodies (turn off hood light) and let sit for 20 min at 4°C (or 10-15 min at RT) in the dark (wrap in aluminium foil)
        • We use EpCAM and CD49f to separate cells into luminal (EpCAMhi/CD49flo) and basal (EpCAMlo/CD49fhi) populations – stromal cells will be negative for both
    • Wash x1-2 with 3 mL PBS + 2% CS per tube
    • Resuspend samples in 200 uL PBS + 2% CS
    • Keep on ice and in dark until analysis