Anti cd16 32 clone 93

The upper left lung lobe was removed and cut into small pieces with a razor. The lung tissue was then transferred to a C-tube (Miltenyi Biotec, Auburn, CA) and processed using digestion buffer containing 1mg/ml of Collagenase D and 0.1 mg/ml DNase I (Roche, Indianapolis, IN) in HBSS and a GentleMACS dissociator (Miltenyi Biotec), according to manufacturer's instructions. The homogenates were then filtered through 70 um nylon cell strainers to obtain a single cell suspension. Red blood cells were lysed using ACK lysis buffer (Life Technologies, Grand Island, NY). Cells were counted using trypan blue to exclude dead cells. To assess neutrophil numbers, 1×10⁶ lung cells were first incubated with anti-CD16/32 (clone 93,eBioscience, San Diego, CA) to block unspecific binding to the Fcy II/III receptor. Cells were then immunostained with rat anti-mouse antibodies: CD45 e780 (clone30-F11, eBioscience), CD11b e450 (clone M1/70, eBioscience), and Ly6G (Gr-1) PE Cy7-conjugated (clone RB6-8C5, eBioscience). Antibody incubation was carried out for 30 minutes at 4°C. Cells were washed and fixed as described (Boehmer, Goral, Faunce, & Kovacs, 2004 (link); Murdoch et al., 2011 (link)). Samples were run on a BD Fortessa cytometer (BD Biosciences, San Jose, CA). Data analysis was performed using Flow Jo FCS analysis software (Tree Star Inc., Ashland, OR).

The upper left lung lobe was removed and cut into small pieces with a razor blade. The lung tissue was then transferred to a C-tube (Miltenyi Biotec, Auburn, CA) and processed using digestion buffer containing 1mg/ml of Collagenase D and 0.1 mg/ml DNase I (Roche, Indianapolis, IN) in HBSS and a GentleMACS dissociator (Miltenyi Biotec), according to manufacturer’s instructions [28 (link)]. The homogenates were then filtered through 70 um nylon cell strainers to obtain a single cell suspension. Red blood cells were lysed using ACK lysis buffer (Life Technologies). Cells were counted using trypan blue to exclude dead cells. To assess mesenchymal stem cells, 1×10⁶ lung cells were first incubated with anti-CD16/32 (clone 93, eBioscience, San Diego, CA) to block unspecific binding to the Fcy II/III receptor. Cells were then incubated with rat anti-mouse F4/80 APC (clone BM8, eBioscience), CD11b eFluor 450 (clone M1/70, eBioscience), CD11c APC-eFluor 780 (clone N418, eBioscience), and Siglec-F PE-CF594 (clone E50-2440, BD Biosciences, San Jose, CA). Antibody incubation was carried out for 30 minutes at 4°C. Cells were washed and fixed as described [56 (link), 57 (link)]. Samples were run on a BD Fortessa cytometer (BD Biosciences). Data analysis was performed using Flow Jo FCS analysis software (Tree Star Inc., Ashland, OR).

The spleens were harvested and grinded using the rubber tip of the syringe, after which the red blood cells were removed by ACK lysis buffer. The cells were filtered through nylon mesh filters and washed with PBS. The single-cell suspensions were incubated with anti-CD16/32 (clone 93; eBioscience) to reduce non-specific binding to Fc receptors (FcRs). For analysis of the activated DCs, cells were stained with anti-mouse CD11c-APC and anti-mouse CD86-FITC antibodies. For analysis of active T cells in the spleens, cells were stained with Live/Dead-BV510, anti-mouse CD3-AF700, anti-mouse CD4-APC, and anti-mouse CD8a-FITC. The Stratedigm S1200Ex flow cytometer (Stratedigm) was used for flow cytometry, and data analysis was performed using FlowJo software.

Spleens and LNs were mashed through a 70 μm cell strainer (BD Biosciences) using the back of a syringe plunger. Tumors were mechanically dissociated by dicing into small (~1–2mm) pieces using a scalpel and then mashed through a 70 μm cell strainer using the back of a syringe plunger. Cell suspensions were resuspended in complete RPMI (cRPMI; RPMI [Invitrogen] plus 2 mM L-glutamine, 1 mM sodium pyruvate, pen/strep [50 μg/ml], and 10% FCS) and passed through a 70 μm cell strainer. Cells were washed with PBS, and red blood cells in tumor and spleen pellets were lysed using RBC lysis buffer (Biolegend). Cells were washed with PBS, stained using LIVE/DEAD Aqua (Invitrogen), then washed and Fc receptors blocked with anti-CD16/32 (clone 93; eBioscience) before staining with surface antibodies (listed in Supplementary Table S1).
For intracellular TNF analysis, cells were stimulated in 24-well plates with 20 nM PMA (Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich) at 37°C for 4 hours. After 1 hour, GolgiStop (1μl/ml; BD Biosciences) was added. Cells were stained for surface markers and then TNF following fixation/permeabilization following the manufacturer’s protocol (Foxp3-staining kit; eBiosciences). Data were acquired on a FACS Canto II (BD Biosciences) and analyzed using FlowJo (TreeStar, USA).

Tumors were harvested, treated with 1 mg/mL collagenase I (Gibco, USA) for 1 h, and ground by the rubber end of a syringe. Cells were filtered through nylon mesh filters and washed with PBS. The single-cell suspension was incubated with anti-CD16/32 (clone 93; eBiosciences) to reduce nonspecific binding to FcRs. Cells were further stained with the following fluorochrome-conjugated antibodies: CD45 (30-F11), CD3e (145-2C11), CD4 (GK1.5), CD8 (53-6.7), Foxp3 (FJK-16s), CD11b (M1/70), Ly6C (HK1.4), Ly6G (RB6-8C5), F4/80 (BM8), B220 (RA3-6B2), NKp46 (29A1.4), and PI staining solution (all from eBioscience). LSR FORTESSA (BD Biosciences) was used for cell acquisition, and data analysis was carried out using FlowJo software (TreeStar, Ashland, OR).