70 μm nylon mesh filter

Adenoids and tonsils were surgically removed, immediately placed in R10 media [RPMI-1640 (Lonza, CA, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, MA, United States), 100 U/mL Penicillin/Streptomycin (Gibco, MA, United States), and 1% L-Glutamine (Gibco, MA, United States)] and transported to the laboratory for processing within 30 min. Tissues were cut manually into small pieces and disaggregated by mechanical disruption using a 70 μM nylon mesh filter (BD Biosciences, San José, CA, United States) to obtain a single-cell suspension. Disaggregated cells were washed twice in R10, and red blood cells were lysed using CK (Chloride-Potassium) lysis buffer (Lonza, MD, United States). Single-cell suspensions were manually counted and washed with phosphate-buffered saline (PBS, Lonza, CA, United States) prior to being subjected to flow cytometry-based immunophenotyping.

Tissue dissociation and SP isolation were done as described previously.^{8 (link)} The samples stored in liquid nitrogen were thawed at 37 °C and washed with Hank’s balanced salt solution (Life Technologies, UK). After dissociation using Liberase Blendzyme 3 (Roche, Basel, Switzerland) at a concentration of 0.8 Wunsch unit/ml during 1 h at 37 °C, the samples were filtered with a 70 μm nylon mesh filter (BD Biosciences, Erembodegem, Belgium) and gradient centrifugation based on 15% Percoll/Hank’s balanced salt solution (100 × g for 15 min). Finally, the pelleted cells were resuspended in HepatoZYME-SFM (Life Technologies, UK) with 1% penicillin/streptomycin (Life Technologies, UK) added to obtain a single-cell suspension.

EG7 cells were injected subcutaneously. Eighteen hours later, to detect DCs in the skin, skin tissues were excised, cut into small pieces and digested with DNase I (0.1 mg/ml; Sigma) and collagenase Type IV (4 mg/ml; Gibco) for 2 h at 37 °C. The resulting cell suspensions were centrifuged, washed in PBS and passed through a 70-μm nylon mesh filter (BD Biosciences) to obtain single-cell suspensions. The single-cell suspensions were stained with PerCP-Cy^TM5.5-conjugated anti-CD45, PE-conjugated anti-MHC-II, APC-conjugated anti-CD11c (1:100; BD Biosciences) and FITC-conjugated anti-IFN-β (1:100; PBL Assay Science) antibodies for a flow cytometry assay.

Preparation of spleen lymphocytes was performed as previously reported44 (link). Briefly, the immunized or control mice were killed on day 7 after the third immunization. Single-cell suspensions were obtained from spleens through a 70-μm nylon mesh filter (BD Biosciences), and lymphocytes were enriched by lymphocyte separation medium (Dakewe Biotech Company) according to the manufacturer’s instructions. To assess the efficacy of T cells in antitumor in vivo, spleen lymphocytes (1 × 10⁷) from donor mice on day 7 after the third immunization were adoptively transferred intravenously into recipient mice one day before and three days after the tumor inoculation (3 × 10⁶). Immunoglobulins were purified from the pooled sera derived from the immunized or control mice by affinity chromatography (CM Affi-gel Blue Gel Kit; Bio-Rad). To assess the efficacy of Immunoglobulins in antitumor activity in vivo, the purified Immunoglobulins (50 mg/kg) were adoptively transferred intravenously one day before mice were challenged with 3 × 10⁶ tumor cells and then treated twice per week for 3 weeks.

Spleens were harvested from female C57BL/6 mice (4–6 weeks old) that were previously immunized with XPa one week before being sterilized. Cell suspensions were generated by filtration through a 70-μm nylon-mesh filter (BD Biosciences), and lymphocytes were enriched via specific separation medium and density-gradient centrifugation and isolated with an EasySep Mouse T cell Isolation Kit (19851, STEMCELL Technologies). Then, T cells were stained with 1 mM carboxyfluorescein (CFSE, 423801, Biolegend Inc., San Diego, CA, USA), and CFSE-stained T cells were cocultured with DCs treated with saline, LPS (100 ng/mL; Sigma L9143), XPa (10 MOI), XPa sup (same volume of XPa), XPa + DNase I (10 MOI), or CD3/CD28 (Dynabeads Mouse T-Activator CD3/CD28, Gibco, 11452D) at a DC:T-cell ratio of 1:10. After 72 h of coculture, cells were collected for flow cytometry analysis, and culture supernatants were collected for cytokine IFN-γ assays.