TIP were isolated as previously described (Chong et al., 2011). Briefly, hearts were minced and incubated in collagenase type II (Worthington, USA) at 37°C before filtering through 40 μm strainers. Cells were resuspended in red cell lysis buffer, followed by dead cell removal, immunostaining for 15 min on ice with fluorophore-conjugated antibodies and two times wash with FACS buffer (1x PBS containing 2% fetal bovin serum) before acquisition.

We employed very stringent gating strategies to exclude doublets in the FACS analysis: FSC-H vs FSC-A, FSC-H vs FSC-W and SSC-H vs SSC-W cytograms were used to discriminate and gate out doublets/cell aggregates during sorting or from the analysis (Figure 1—figure supplement 5). To account for the autofluorescence generated by MI, we used a wild-type MI mouse as control to set up the gating strategy (Figure 1—figure supplement 6).

For the TIP fraction, total DAPI-negative live single cells were sorted in FACS buffer. For the GFP+ fraction, GFP+CD31-cells were sorted from the DAPI-negative live single cells. For Fluidigm experiments, SCA1+PDGFRα+CD31-(S+P+) cells were isolated and sorted as described above. For each sample, at least 10,000 final gate events were collected and stored for the later analyses.

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Q&A
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Andrew Masoud
University of Alberta 
Dear Authors on this great research article,
This is to, kindly, ask for your expert advice on the efficient isolation of Murine Cardiac lymphocytes and microvascular ECs for single cell RNA sequencing purposes.
Being a leading expert along with your research team members, I would appreciate your kind guidance in this regard.
All of the present protocols in the literature are about digestion with Collagenases + other enzymes for 30 to 60 minutes in 37 C.
However, I am abit worried if I digest too much I would destroy cell surface marker antigens or kill the cells, and if too little, I won't get an adequate cell yield.
Thanks for your kind support.
This is highly appreciated.
Sincere regards, Andrew Masoud
2020-07-16 09:34:23 Reply



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