CD45 Monoclonal Antibody (30-F11), PE-Cyanine7, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For cell-surface antigen staining, the samples were incubated with antibodies for 20 min at 4 °C, washed and resuspended in PBS containing 3% FBS. To exclude dead cells, 7-amino actinomycin D (7-AAD, BD Biosciences) was added to the samples before sorting.	We sorted live, single cells using a FACSAria III Cell Sorter with FACSDiva ver. 8.0.1 (BD Biosciences).

Protocol tips
For cell-surface antigen staining, the samples were incubated with antibodies for 20 min at 4 °C, washed and resuspended in PBS containing 3% FBS. To exclude dead cells, 7-amino actinomycin D (7-AAD, BD Biosciences) was added to the samples before sorting.
Downstream tips
We sorted live, single cells using a FACSAria III Cell Sorter with FACSDiva ver. 8.0.1 (BD Biosciences).

Publication protocol

For cell-surface antigen staining, the samples were incubated with antibodies for 20 min at 4 °C, washed and resuspended in PBS containing 3% FBS. To exclude dead cells, 7-amino actinomycin D (7-AAD, BD Biosciences) was added to the samples before sorting. We sorted live, single cells using a FACSAria III Cell Sorter with FACSDiva ver. 8.0.1 (BD Biosciences). Sorted cells were collected in DMEM containing 10% FBS, antibiotics, antimycotic solution, and 25 mM HEPES for further analyses. For proSP-C staining, single cell suspension of the lung or the sorted cells were incubated with fixable viability dye eFluor780 (eBioscience) and surface antigens, fixed, and permeabilized with fixation and permeabilization buffer (eBioscience) according to the manufacturer’s instructions. Permeabilized cells were incubated with anti-mouse proSP-C antibody or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and labeled with PE-conjugated F(ab’)2 donkey anti-rabbit IgG (1:200; eBioscience). Fixed cells were analyzed using a BD LSR Fortessa (BD Biosciences). Appropriate isotype control samples were utilized for all FACS analyses. The data were analyzed using FlowJo software (ver. 7.6.5, Tree Star, San Carlos, CA).

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