Protocols
Heart Flow Cytometry Protocol
Euthanize mouse and sterilize with 70% ethanol.
Remove hair, skin, muscle, and open the thoracic cavity to expose the heart.
Using a 26-gauge needle inserted into the left ventricle, perfuse heart with 20 mL cold PBS to remove blood and peripheral cells.
Excise heart or region of interests and store in cold PBS in a microfuge tube on ice. (Optional: If absolute cell numbers per milligram of tissue is desired, obtain the wet weight of the heart by placing on a piece of weigh paper at tare weight).
Prepare 1 mL/heart Digestion Buffer (Collagenase type II 600U/mL, Hyaluronidase 60U/mL, DNase 0.01mg/mL) in HBSS.
Remove PBS and add 1 mL Digestion Buffer to each heart in microfuge tube.
Using scissors, finely mince the heart in Digestion Buffer.
Incubate digesting hearts at 37°C with agitation/rotation for 30 minutes (Optional: Mince hearts every ~10 minutes to ensure complete digestion).
After incubation, transfer digested heart to a 40mM filter inserted into a 6-well plate containing 3 mL cold PBS in the well.
Using the flat end of a 1 mL syringe, carefully triturate the heart through the 40mM filter.
Transfer the single-cell suspension to a 15 mL centrifuge tube.
Centrifuge at 2000 rpm for 5 minutes at 4°C.
Discard supernatant and resuspend the pellet in 1 mL of RBC Lysis Buffer (BioLegend RBC Lysis Buffer Cat#420301 is provided as a 10X solution. Prepare a 1X solution using DI water).
Incubate in RBC Lysis Buffer for 1 minute at room temperature and neutralize the reaction by adding 4 mL cold Stain Wash Buffer (SWB is PBS with 2% FBS and 2mM EDTA).
Centrifuge at 2000 rpm for 5 minutes at 4°C.
Discard supernatant and resuspend in 1 mL TruStain FcX Block in SWB (1:100 dilution TruStain FcX Block-BioLegend Cat# 101319).
Optional: Count viable cells on a hemocytometer using a small aliquot of sample, ~50 mL, and trypan blue solution, ~50 mL, to obtain an absolute number of cells.
Optional: Transfer samples to microfuge tube or 96-well plate. Plate enables simultaneous staining of large number of samples. Volumes should be adjusted accordingly for tube or plate.
Incubate in TruStain FcX Block for 10 minutes on ice (or 4°C) to block non-specific binding of antibodies. After incubation, add 4 mL cold PBS.
Centrifuge at 2000 rpm for 5 minutes at 4°C.
Discard supernatant and resuspend sample in 200 mL of PBS with BioLegend Zombie Aqua Viability Dye (0.1-1mL per sample, Cat#423101 located in 20°C).
Incubate at room temperature in the dark for 15 minutes.
Neutralize the reaction by adding 4.8 mL of SWB.
Centrifuge at 2000 rpm for 5 minutes at 4°C.
Discard supernatant and resuspend sample in 200 mL of SWB containing primary antibodies (frequent dilution is 1:200 and specific antibodies will depend on populations of interest).
Incubate on ice (or 4°C) in the dark for 20 minutes.
After incubation, add 4.8 mL of SWB.
Centrifuge at 2000 rpm for 5 minutes at 4°C.
Discard supernatant and resuspend in 5 mL of SWB.
Centrifuge at 2000 rpm for 5 minutes at 4°C and discard supernatant.
If analyzing samples immediately, resuspend in 500-1000 μL SWB and store on ice or at 4°C.
If analyzing samples the next day, resuspend in 500-1000mL containing equal parts SWB and 2% formaldehyde in PBS and store at 4°C..