Biotinylated anti cd45

The cells were incubated with biotinylated anti-CD32 (0.5 µg/million cells; BD Pharmingen, San Diego CA, USA) and biotinylated anti-CD45 (1.5 µg/million cells; BD Pharmingen) for 30 min at 37 °C. Meanwhile, streptavidin-coated magnetic particles (Thermo Fisher Scientific, Waltham, MA, USA) were washed twice in phosphate-buffered saline (PBS) (10 min each wash) in polypropylene culture tubes using a magnetic tube separator (Sigma-Aldrich). After incubation, the cells were centrifuged (130 × g for 8 min at 4 °C), resuspended in 7 mL of DMEM, added to the magnetic particles, and incubated with gentle rocking for 30 min at room temperature. At the end of the incubation, the tubes were attached to the magnetic tube separator with adhesive tape for 15 min. The cell suspension was aspirated from the bottom of the tube using a narrow-stemmed transfer pipette, centrifuged, and resuspended in a culture medium. Cell viability was determined by trypan blue staining. The purity of alveolar type II cells was assessed via pro-SPC immunofluorescence staining. As in previous studies, 4–8 × 10⁵ cells were isolated from a single mouse. In these samples, the purity of type II pneumocytes was 90–93%. The isolated cells were maintained in 10% FBS and 1% penicillin–streptomycin in 25 mM HEPES-buffered DMEM.

The cells were incubated with biotinylated anti-CD32 (0.5 µg/million cells; BD Pharmingen) and biotinylated anti-CD45 (1.5 µg/10⁶ cells; BD Pharmingen) for 30 min at 37 °C. Streptavidin-coated magnetic particles (Thermo Fisher Scientific, Waltham, MA, USA) were washed twice in phosphate-buffered saline (PBS) (10 min per wash) in polypropylene culture tubes using a magnetic tube separator (Sigma-Aldrich). After incubation, the cells were centrifuged (130 × g for 8 min at 4 °C), resuspended in 7 mL DMEM, added to the magnetic particles, and incubated with gentle rocking for 30 min at room temperature. At the end of incubation, the tubes were attached to the magnetic tube separator with adhesive tape for 15 min. The cell suspension was aspirated from the bottom of the tube using a narrow-stemmed transfer pipette, centrifuged, and resuspended in culture medium. Cell viability was determined by trypan blue staining. The purity of type II AECs was assessed by pro-SPC immunofluorescence staining. As in previous studies, 4–8 × 10⁵ cells were isolated from a single mouse. In these samples, the purity of type II pneumocytes was 90%–93%. Isolated cells were maintained in 10% FBS and 1% penicillin–streptomycin in 25 mM HEPES-buffered DMEM.