40 μm nylon cell strainer

Peritoneal macrophages were isolated from C57BL/6 mice [23 (link)]. Macrophages were treated with 1 μg/mL of LPS for 4 hr prior to treating overnight with 2 μg/mL of LT-IIb, LT-IIc, LT-IIbc, or LT-IIcb. Culture supernatants were collected the following day and stored at -80°C until analyzed by ELISA. Treated macrophages were detached from the cell culture wells with enzyme-free EDTA disassociation buffer (Sigma) and mechanically disrupted with cell scrapers (Corning, Tewksbury MA). Cells were filtered through 40 μm nylon cell strainer (Corning) prior to staining for flow cytometry.

For the FDM mouse model, 21-day-old C57BL/6J mice with diffuser goggles were used (Schaeffel et al., 2004 (link); Wu et al., 2018 (link)). After 2 days’ form deprivation treatment, the mice were sacrificed by cervical dislocation, with their eyeball enucleated and dissected to obtain the whole retina. The retinas were digested in collagenase IV (6 mg/mL; Gibco BRL, Grand Island, NY, United States) for 1 min, before moving the RPE layer. Digestion in papain solution (10 mg/mL Dulbecco phosphate-buffered saline (DPBS; Sigma-Aldrich) of remaining retina was performed for a further 30 to 45 min. Papain was then neutralized with a trypsin inhibitor solution (0.15% ovomucoid in DPBS; Gibco BRL), and the tissue was triturated with 200-μL pipette tips to generate a single-cell suspension. The cells were pelleted, resuspended, and filtered through a 40-μm nylon cell strainer (Corning Inc., Corning, NY, United States) to eliminate all clumped cells. The cells were then diluted in DPBS to ∼1,000 cells/μL for use.

E13.5 lung lobes were harvested from control and Znhit1 cKO embryos to isolate epithelial cells. Dispase (Corning) was used for lobe digestion. After 15 min of digestion at 37 °C, samples were transferred to 5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 25 mM Hepes (Gibco) and 120 units of DNAse I (Sigma–Aldrich). Sample dissociation was performed on GentleMACS Dissociator (Miltenyi Biotec), and 40 μm nylon cell strainers (Corning) were used to get single cells. Cell suspensions that passed through the strainers were pelleted by centrifugation at 1500 rpm (433g) for 6 min at 4 °C and resuspended in 10 ml DMEM (with Hepes and PenStrep) and centrifuged once again. Cells were resuspended in 90 μl of autoMACS Running Buffer (Miltenyi Biotec) and 10 μl FcR Blocking Reagent and incubated at 4 °C for 10 min. Then, 10 μl of CD326 MicroBeads (Miltenyi Biotec) was added to bind epithelial cells. After incubation for 15 min at 4 °C, cells were washed twice with autoMACS Running Buffer. Cell pellets were resuspended in 500 μl of Running Buffer to pass through a 40 μm nylon filter before sorting using an AutoMACS Pro Separator (Miltenyi Biotec). CD326-positive (epithelial) and -negative (nonepithelial) cells were separated. Sorting efficiency was determined by the qRT–PCR of Epcam (epithelial) and Twist2 (mesenchymal).