70 μm cell strainer

Mouse crypt isolation and organoid establishment were performed as previously described [22 (link)]. Briefly, the jejunum fragments were incubated in EDTA (2 mM) with PBS for 30 min on ice. After removing EDTA solution, the jejunum fragments were gently washed with cold PBS. The crypt-villus was collected through scraping the inner side of intestine and resuspended in PBS. After standing for 5 min, the sediment was retained and passed through a 70 μm cell strainer (Biosharp, Hefei, China). The isolated crypts were centrifuged at 65×g for 5 min and resuspended in matrigel (Corning, Glendale, AZ, USA) for culture and formation of organoids. For irradiation treatment, organoids were seeded into 48-well plates at a density of ∼100 organoids/20 μL matrigel per well.
Organoid survival assays were performed as described previously [23 (link)]. Briefly, the number of live/dead organoids in each group was counted on day 9 after IR, and the ratio of organoid survival was calculated. Live organoids fulfil the following criteria: the diameter of organoid >30 μm, with solid or cystic structure. The dead organoids were identified by the characteristics, the structural collapse and the presence of dispersed cells or dark debris where the organoid was previously located. The sizes of live organoids were measured with the software ImageJ (National Institutes of Health).

Chicken peripheral blood mononuclear cells (PBMCs) were prepared as our previous report (He et al., 2022 ). Single-cell suspensions from tissues were also isolated with a separation kit for chicken tissue (TBD, Tianjin, China). Briefly, spleens or thymuses were mechanically disrupted and pushed through 70-μm cell strainers (Biosharp, Hefei, China) with the plungers of 5 mL syringe. The cells were resuspended in 6 mL phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS) (Gibco, Grand Island, NY), and then overlaid onto the separation solution at a 1:1 ratio and centrifuged at 500 × g for 30 min at room temperature (RT). The interface containing mononuclear cells were collected, washed twice, and then resuspended in complete RPMI1640 medium containing 5% FBS, 5% chicken serum (Gibco, Grand Island, NY), penicillin (100 U/mL), and streptomycin (0.1 mg/mL) (Beyotime, Shanghai, China).