Facs caliber apparatus

NHEJ and HR assays were previously described [19] (link). Briefly, to express I-SceI, pCMV-NLS-I-SceI was introduced by transfection, using Lipofectamine 2000 reagent, into 1.5–3×10⁵ H1299 dA3-1#1 cells (for NHEJ) [25] (link) or 3×10⁵ HeLa pDR-GFP cells (for HR) [26] (link) pretransfected with siRNA for 48 hr using Lipofectamine RNAiMAX (Invitrogen). siBRM for the NHEJ assay or siBRCA1 for the HR assay was used as a positive control [25] (link). EGFP-positive cells were counted with Cellquest software. For FACS analysis, cells were harvested by trypsinization, washed with PBS, stained, and applied on the FACS caliber apparatus (Becton Dickinson).

Peritoneal macrophages were isolated from 7-week-old IL-13Rα2 null, TMEM219 null and WT mice, which had been injected intraperitoneally 3 days earlier with 3 ml of thioglycollate. The collected cells were washed twice with PBS and then cultured in DMEM containing 10% heat-inactivated FBS, 1 mM glutamine, 100 of IU ml⁻¹ penicillin and 0.1 mg ml⁻¹ of streptomycin at a density 2 × 10⁶ cells per ml. The cells were then allowed to adhere for 3 h to a 24-well culture flask at 37 °C in a 5% CO₂ incubator. Then, the cultures were washed twice with PBS to remove non-adherent cells before the addition of 1 ml of fresh medium. Antibodies against F4/80 (565410, PharMingen, San Diego, CA, USA) and FACS analysis on a FACSCaliber apparatus (Becton Dickinson, San Jose, CA, USA) were used to assess cellular purity. In select experiments macrophages were incubated with IL-13, rChi3l1 or vehicle control. Experiments with rChi3l1 were done in the absence of serum.

NHEJ and HR assays were previously described. Briefly, to express I‐SceI, pCMV‐NLS‐I‐SceI was introduced by transfection, using Lipofectamine 2000 reagent (Invitrogen), into U2OS‐EJ5‐GFP cells (for NHEJ) or U2Os‐DR‐GFP cells (for HR) pre‐transfected with KLF4 or ShKLF4 for 48 h using Lipofectamine 2000 (Invitrogen). EGFP‐positive cells were counted with Cellquest software. For FACS analysis, cells were harvested by trypsinization, washed with PBS, stained, and applied on the FACS caliber apparatus (Becton Dickinson).