Hanks Balanced Salt Solution

Colon LP cells were isolated as described previously and stained for flow cytometry (20 (link)). Briefly, the whole colon was washed, cut into 1-1.5 cm sections, incubated twice in Hanks' Balanced Salt Solution (HBSS, Sigma-Aldrich, St. Louis, MO) with 5mM EDTA at 37°C and then digested in RPMI-1640 containing 1 mg/ml collagenase type 1 (Worthington, Lakewood, NJ) and 10% FBS at 37°C for 1h in a shaking incubator. The cells were collected from the interface of 40/80% Percoll gradients (Sigma-Aldrich). The fluorescent dye conjugated-antibodies listed below were used for flow cytometry: Anti-CD3 (145-2C11), CD4 (GK1.5) (Biolegend, San Diego, CA), Thy1.2 (30-H12), CD45.2 (104) (BD Biosciences, San Jose, CA), FoxP3 (FJK-16s), RORγt (B2D), IL-22 (IL22JOP), IL-17A (TC11-18H10) (eBioscience, San Diego, CA). For intracellular cytokine staining, cells were stimulated with PMA (0.1 μg/ml, Sigma-Aldrich), ionomycin (0.5 μg/ml, Sigma-Aldrich) in the presence of Brefeldin A (10 μg/ml, Sigma-Aldrich) for 4 h. For IL-22 intracellular staining, mouse recombinant IL-23 (40 ng/ml, R&D systems, Minneapolis, MN) was added to the PMA, ionomycin and Brefeldin A cultures for 4 h. The cells were fixed and permeabilized using kits for intracellular staining (eBioscience) according to the manufacturer's instructions. All data were collected on a BD Fortessa LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).

The whole blood of mice was collected with an anticoagulant tube, and neutrophils were extracted using a mouse peripheral blood neutrophil isolation kit (Solarbio, Beijing, China). The concentration of extracted cells was counted by cell counter. Measurement of ROS levels was determined as previously described with minor modifications (Wu et al., 2009 (link)). Neutrophil cells were diluted to 10⁵/ml using warm Hanks’ balanced salt solution (HBSS) containing 100 mM luminol and 1 U/ml horseradish peroxidase. The diluted cell suspension was added to a 96-well plate at 200 μl/well, and the reaction was performed at 37°C for 10 min. The bacterial strains were added at a multiplicity of infection (MOI) of 5. At the same time, 10⁶ cells were removed and infected with an MOI of 10. The reaction was carried out in a cell incubator at 37°C, and 20 μl reactant was removed every hour for dilution and dropping plate counting, to which 100 mM NAC was added if necessary.

The testes were washed with Hanks Balanced Salt Solution (HBSS) three times and digested in 2 mL of GEXSCOPETM Tissue Dissociation Solution (Singleron Biotechnologies) using a Singleron PythoN™ Automated Tissue Dissociation System (Singleron Biotechnologies) at 28 °C for 15 min. The mixture was then centrifuged at 500 g for 5 min and resuspended with PBS. Finally, the samples were stained with trypan blue (Sigma-Aldrich) and cellular viability was evaluated microscopically.

For a large-pooled screen, viral production and functional titration were conducted in the same manner as described previously (Wang et al., 2020 (link)). Pooled human GeCKOv2 plasmids (lentiCRISPRv2) were cotransfected with the lentiviral packaging plasmids pLP1, pLP2, and pLP/VSVG (Thermo Fisher Scientific) into Lenti-X 293T cells (Clontech). 12 h later, the medium was changed to 10 ml prewarmed DMEM supplemented with 10% FBS. The viral media was collected 24, 48, and 72 h after transfection and filtered through a membrane (Millex 0.45 µm, poly vinylidene di-fluoride, 33 mm). Finally, 30 ml viral media in total was combined and stored at 4°C for functional titration and pooled screening as quickly as possible.
PIGS-HRD1-DKO cells were plated in 8 × 15 cm dishes (3.5 × 10⁶ cells per dish). Approximately, 8 × 10⁷ cells (1 × 10⁷ cells per dish) were transduced with viral supernatant after 36 h of seeding. Cells were selected with 0.5 μg ml⁻¹ puromycin until the infected cells were expanded to 2.4 × 10⁸ to maintain the complexity of the gRNA library. Cells were combined and split 1:4, and a minimum of 6 × 10⁷ cells were plated for culture. At 2 wk after transduction, a pellet of 5 × 10⁷ cells without sorting was stored at −80°C. For cell sorting, ∼1 × 10⁸ cells were harvested and incubated with T5 mAb, followed by staining with anti-mouse IgM. After washing with PBS, the cells were resuspended in Hanks’ Balanced Salt Solution (H6648; Sigma-Aldrich), and T5 mAb staining-negative cells were sorted by FACSAria (BD). We prepared 2 × 10⁷ cells for the second sorting. After sorting, the cells were maintained in DMEM supplemented with 0.25 μg ml⁻¹ puromycin. Pellets of 2 × 10⁷ sort2 cells were stored at −80°C until use. For analysis, cells were cultured in 6-well plates 1 d before analysis. The cells were harvested and washed once with PBS and then stained with CD59 or T5 mAb in FACS solution (PBS containing 1% BSA and 0.1% NaN₃) on ice for 25 min. They were then washed twice in FACS buffer, followed by staining with Alexa Fluor 647–conjugated goat anti-mouse IgM or PE-conjugated goat anti-mouse IgG. After two washes with FACS buffer, the cells were analyzed using a BD FACSCanto II.