Primary mouse EC isolation

Night Before

Prepare Dynabead (sheep anti Rat IgG, at# 11035) +αCD31 (Rat anti mouse CD31, BD # 553370) antibody solution. 3-5 uL beads per mouse, and 0.12 uL antibody per dynabead.
            - wash beads with 0.1% BSA (50 mg BSA/50 mL PBS, or 500 mg/500 mL) 3x. Use magnetic dynabar.  
            - overnight incubation should be done in 0.5 to 1 mL 0.1% BSA.

On Bench

1. Prepare enzyme solution and keep at 4C until time of use.
   1. For heart, lung: 2 mg/mL Collagenase I (Stem Cell Technologies Cat#07416) in serum free DMEM. 6 mL per mouse. Example: if collecting from 4 mice total, use 24 mL DMEM and 48 mg Collagenase I.
   2. For liver (whole organ): 2 mg/mL Collagenase I in serum free DMEM. 10 mL per mouse.
   3. For skeletal muscle: 2 mg/mL Collagenase I and 1mg/mL Dispase II (Sigma Cat#D4693) in serum free DMEM.
2. Sac mice with approved euthanasia protocol
3. Optional: Perfuse animals 10-15 mL cold PBS/saline to remove blood
4. Skin mouse and remove organ(s) of interest. Briefly wash in (cold) PBS/HBSS and keep in cold, serum free DMEM. Keep tissue on ice at all times.
   1. Cut tissue in an Eppendorf tube, using small scissors to snip
   2. For heart, lung, and liver, cut into ~1 mm² pieces.
   3. If skeletal muscle, might require finer mincing. Make sure to remove fat and fascia.
5. Add tissue to Falcon tube containing enzyme solution.
6. Put in 37 C shaking incubator (150 RPM) for 15-30 minutes.

Move into Tissue Culture Sterile Hood

1. Triturate each sample with serological pipette to break up remaining large tissue chunks.
   1. May need to use cannula if large chunks remain (Liver isolation, for example).
2. Pour through 100-micron filter into 50 mL.
3. Gently mash the tissue chunks in the filter with 1 or 10 mL syringe plunger.
4. Pour 10 mL of cold DMEM (10% FBS) through filter.
5. Repeat 8-9.
6. Pour filtrate into new 50 mL and strain using 40-micron filter this time.
7. Add 10 mL cold DMEM (10% FBS) through filter.
8. Spin at 200-300g for 4-5 minutes. Remove supernatant carefully.
9. Wash with cold 0.1% BSA, spin again.
10. Meanwhile, wash dynabead+αCD31 solution 3x with BSA as before (removes unbound αCD31).

CD31+ cell selection

1. Resuspend each cell pellet in 1 mL of the dynabead+αCD31 solution. Incubate on rotator for 10 minutes at room temperature or 15 minutes at 4 C.
   1. If cell pellet is too large, may need to incubate in larger volume. For example, 4 mL for liver EC.
2. Mount on magnetic bar and wash beads+cells with 0.1% BSA 4-6x. Triturate vigorously.
3. For qPCR analysis: Resuspend each pulldown into lysis buffer of your choosing.
4. For cell culture: Resuspend in growth media and add to gelatin-coated tissue culture plate.
5. Refresh media after two days and visually assess yield.

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