70 μm nylon cell strainer

Two surgery removed early-stage HCC tumors (labeled as “HCC-1/-2”) were obtained from the inform-consent HCC patients (All male, 47 and 55 years old), hospitalized at authors’ institutions. The HCC tissues were washed and mechanically dissociated [31 (link)], which were then subjected to digestion via incubation in triple enzyme medium (1 × collagenase, 1 × hyaluronidase, and 1 × DNase) at room temperature for 1 hour. The primary cells were then filtered through a 70-μm nylon cell strainer (Becton Dickinson, Shanghai, China) and suspended in complete medium for primary cells [31 (link)]. The protocols requiring clinical samples were approved by the Ethics Review Board (ERB) of all authors institutions, and were in line with the principles expressed in the Declaration of Helsinki.

Primary bone marrow-derived MSCs were obtained as described previously [7 (link)]. Briefly, bone-marrow cells were flushed from the femur and tibia of SJL mice and passed through a 70-μm nylon cell strainer (Becton–Dickinson, Franklin Lakes, NJ, USA). The cell suspension was seeded in the presence of Murine Mesencult as medium (Stem Cell Technologies, Cambridge, UK) and MSCs were expanded and tested after 8 passages. They were identified as MSCs according to their expression of CD9, SCA-1 and CD44 and their lack of expression of CD45 and MHC II markers, assessed using flow cytometry with appropriate conjugated monoclonal antibodies: FITC anti-CD9, PE anti-SCA1, APC anti-CD44, PerCp anti-CD45 and PB anti-MHC II (Biolegend, San Diego, CA, USA). MSCs were also tested for their ability to reduce T-cell proliferation [7 (link)].

The ACL remnant tissues were harvested from the rupture site of 1.0 cm³ in size, preserved in the sterile saline solution on ice, minced into 1–2 mm small particles, washed by phosphate-buffered saline (PBS) three times, and then digested with 0.4 % collagenase type II (0.4 % w/v; Worthington Biochemical Corporation, Lakewood, New Jersey) for two hours in a 37 °C 5 % CO₂ incubator. The suspension was washed with PBS and centrifuged at 1500 rpm for 10 min, and the supernatant was discarded; the cells were suspended in PBS, and then filtered through a 70-μM nylon cell strainer (Becton Dickinson [BD], Franklin Lakes, New Jersey). Later, the cells were washed with PBS twice, cultured in standard Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Sigma-Aldrich) at a density of 1 × 10⁵ in 75 cm² collagen-coated flask, then incubated in 37 °C 5 % CO₂ incubator. The medium was changed every 3 days, and passaged the cells when they reached 70–80 % confluent.

To isolate pulmonary leukocytes from naïve mice, the pulmonary vasculature was perfused with 10 ml cold PBS after blood collection via cardiac puncture. The perfused, non-lavaged lungs were then dissociated mechanically with scissors and incubated with Liberase TM (1 mg/ml; Roche, Indianapolis, IN) and DNAase I (1 mg/ml; Sigma Aldrich, St. Louis, MO) for 10 min at 37 °C. Lung digests were filtered through a 70-μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ) and erythrocytes removed using RBC lysis Buffer. Cells were re-suspended in FACS buffer.
In LPS experiments, mice underwent broncho-alveolar lavage prior to lung harvesting. Mice had their non-perfused, lavaged lungs inflated with 10 % formalin, paraffin-embedded, and processed for H&E staining. Lungs (all 5 lobes) were scored for an average of severity of inflammation (0 = none, 1 = mild, 2 = moderate, 3 = severe) and distribution (0 = none; 1 = focal; 2 = multifocal; 3 = extensive). In separate experiments, LPS-treated mice had both vasculature perfusion and broncho-alveolar lavage prior to collection of the lung for mechanical and enzymatic dissociation as described above. Cells from these experiments were processed for FACS as below.

Cells were stained with fluorochrome-labelled antibodies and subsets identified for sorting as described in Fig 1. All incubation and washing steps were performed in sodium azide-free FACS buffer. After a final wash prior to sorting, cells were filtered through a 70μm nylon cell strainer (Becton Dickinson) for removal of cell clumps. Sorted populations were collected in complete medium (sDMEM) for culture.

Samples of blood, endocervical brushings, and endometrial biopsies collected at UCSF were transported on ice to UC Davis to arrive within 3–4 hr of collection (Figs 2, 3, 4). Upon arrival, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll–Hypaque (Pfizer, New York, NY, USA) density gradient centrifugation, and washed in PBS. Mononuclear cells from endocervical brushings were isolated by rubbing the brushes together, pipetting several times, passing through a 70‐μm nylon cell strainer (Becton Dickinson, Bedford, MA, USA) and washing in complete medium [RPMI‐1640 supplemented with 15% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), and glutamine (2 mmol/L)]. Endometrial biopsies were subjected to 2–3 rounds of collagenase type II digestion (0.5 mg/mL; Sigma‐Aldrich, St. Louis, MO, USA), followed by mechanical disruption using an 18‐gauge blunt‐end needle and passage through a 70‐μm nylon cell strainer. The pooled cells were washed in complete medium. Red blood cell lysis was performed as needed on PBMC, endometrium and endocervical cells using ammonium chloride–potassium carbonate–EDTA (ACK).

Spleens were finely minced and passed through a 70-μM nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Spleen cells were collected in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 10% preselected fetal calf serum (FCS), 2 mM l-glutamine (both from Hyclone, Logan, UT), 100 U/mL penicillin, 100 mg/mL streptomycin (both from Life Technologies), and 5 × 10–5 M β-mercaptoethanol (Sigma-Aldrich). Single-cell suspensions were cleared of erythrocytes by hemolysis using a hypotonic buffer (pH 7.2) composed of 0.15 M ammonium chloride, 10 mM potassium bicarbonate (both from Merck, Darmstadt, Germany), and 0.1 mM ethylene-diaminetetraacetic acid (Life Technologies).
To evaluate the composition of APCs, splenocytes were stained for CD3e PerCP-eFluor 710 (clone 17A2, Thermo Fisher Scientific), CD19 FITC (clone 1D3, BD Biosciences), CD11c APC (HL3, BD Biosciences), CD11b PE-Cy7 (clone M1/70, Thermo Fisher Scientific) and HLA-DR BV605 (clone G46-6, BD Biosciences) and analyzed using a BD FACS Aria III (BD Biosciences) and FlowJo software (Version 10.8.1; BD Biosciences). Nonspecific binding through Fc gamma receptors was blocked by a mixture of anti-CD16 and anti-CD32 antibodies (Fc Block; BD Biosciences).