Hepes

The human monocytic leukemia cell line THP-1 from the resource center of Peking Union Medical College Hospital (Beijing, China) and HEK 293T cells from Shanghai stem cell bank (Shanghai, China) were maintained in RPMI 1640 medium (Gibco) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Solarbio), 10 mM HEPES (Biosharp), and 10% fetal bovine serum (Gibco) at 37°C with 5% CO₂ in a humidified atmosphere. Prior to experiments, human monocytes (THP-1) derived macrophages (MΦ-THP-1) were generated by phorbol 12-myristate 13-acetate (PMA, 20 ng/mL) treatment for 48 h followed by resting cells for 12 h according to the methods in references (38 (link)–40 (link)). The mouse macrophage cell line RAW 264.7 was cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 10 mM HEPES, and 10% fetal bovine serum at 37°C with 5% CO₂ in a humidified atmosphere. All cell lines were cultured within 10 passages prior to use. gC1qR knock-down cell line (THP-1-sh-gC1qR) and control cell line (THP-1-sh-luciferase) were prepared as previously described (36 (link)). gC1qR-knockout cell line (THP-1-KO-gC1qR) were generated as described in Materials and Methods below.

The human respiratory epithelial cell line Hep2 (obtained from The Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, China) was cultured in RPMI-1640 medium (Gibco) supplemented with 100 U/ml penicillin and 100 U/ml streptomycin (Solarbio), 10 mM HEPES (AMRESCO0511; Biosharp), and 10% fetal bovine serum (Biological Industries) at 37°C with 5% CO₂ in an incubator.

After epidermal single-cell suspension was prepared, lympholyte-M (Cedarlane Laboratories, Canada) was used to enrich DETCs (purity = 15–20%). For reconstitution assays, DETCs were further purified by the EasySep positive cell isolation systems (PE labeled anti-TCRδ Ab; BD, USA; EasySep mouse PE positive selection kit; StemCell Technologies, Canada). The purity of isolated DETCs was identified by FACS analysis (purity > 90%). Then, DETCs were transferred to wound bed immediately following excision (5 × 10⁴ cells/wound). For in vitro assays, DETC population was expanded in 48-well plates (1–2 × 10⁶/ml) with RPMI containing 10% FBS, 1 μg/ml Con A (Sigma-Aldrich, Germany), 10 ng/ml mouse rIL-2, 1 mM Na Pyruvate (Sigma-Aldrich, Germany), 2 mM glutamine, 25 mM HEPES, 50 μM 2-ME (Biosharp, CN, USA), 100 M non-essential amino acids (Gibco, USA), 100 μg streptomycin and 100 U penicillin for 2–4 weeks. The purity of expanded DETCs (eDETCs) was > 95% by means of FACS analysis. eDETCs were rested without ConA for 2 weeks before used for further experiments.