Gr 1 clone rb6 8c5

Mice were deeply anesthetized with a lethal dose of choral hydrate and transcardially perfused with 0.1 M PBS. Brain was removed and one forebrain hemisphere (without olfactory bulb) was minced, incubated in phenol-red free DMEM supplemented with 2% heat inactivated FBS, 10mM HEPES and Collagenase type IV (0.4 mg/mL) for 15 min and then passed through a 19G blunt syringe to obtain a homogeneous cell suspension. Mononuclear cells were separated with a 40% Percoll gradient. Isolated cells were surface stained in FACS buffer for 20–30 min on ice with the following antibodies: CD11b (clone M1/70, eBioscience), F4/80 (clone CI: A3-1, BioRad), CD45 (clone 30F11, eBioscience), MHC II (clone M5/114.15.2, eBioscience), CD3e (clone 145-2C11, Biolegend), Gr-1 (clone RB6–8C5, Biolegend), CD115 (clone AFS98, eBioscience). Multiparameter analysis was performed on a LSR II Fortessa (BD) and analyzed with FlowJo software (Tree Star) (Details are included in the Flow Cytometry Reporting Summary). Dead cells and doublets were excluded from all analysis.

For these experiments, nanoparticles (CyAL5.5-FH, 10 mg/kg) were injected into the tail vein and mice were sacrificed after a 3-hour incubation. Single-cell suspensions were generated from harvested esophagi following dissociation via rat tail collagenase and trypsin/ethylenediamine-tetraacetic acid (EDTA) digestion. Similarly, spleens were mechanically dissociated and red blood cells were lysed to generate single-cell suspensions of leukocyte populations.^{2 (link)} Staining of cells was performed in PBS + 2% fetal bovine serum. The following antibodies were used for immunophenotyping: CD45 (clone 30-F11), CD19 (clone 6D5), CD3E (clone 145-2C11), CD49b (clone DX5), CD11c (clone N418), F4/80 (clone CI:A3-1), Gr-1 (clone RB6-8C5), and CD11b (clone M1/70), all from BioLegend (San Diego, CA). Nanoparticles were detected via the conjugated CyAL5.5 fluorochrome. Flow cytometry was performed on a BD LSR II instrument (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo version 9.4 software (Tree Star, Ashland, OR). For simulating the modulation of immune cells in response to treatment, 9-month-old L2-Cre;p120^flox/flox mice were treated with dexamethasone (10 mg/kg/day) or PBS (control) once a day for 7 days. Twenty-four hours following treatment, all mice received nanoparticle injection followed by immunophenotyping of CyAL5.5-positive cells as described above.

C57BL/6J and hck^−/−fgr^−/− mice were depleted using an anti-Ly6G mAb (Bio-X-Cell, West Lebanon, NH), as described in ref. 28 (link). Briefly, anti-Ly6G mAb was diluted into sterile endotoxin-free 0.9% NaCl saline solution at a concentration of 1mg/ml. The antibody was injected i.p. at a dose of 0.5 mg per mouse, 17 hours prior to intranasal instillation of LPS (5 µg) or PBS. Control mice were injected i.p. with saline. 2 h after intranasal treatments of depleted and control mice, peripheral blood was collected from all experimental animals to confirm neutrophil depletion by cytofluorimetric analysis. This was performed using a panel of five fluorochrome-conjugated antibodies to CD11b, GR-1 (clone RB6-8C5, Biolegend), CD11c, Ly6C and Ly6G.

Cell surface staining was performed using fluorophore conjugated anti-mouse CD4 clone GK1.5, CD8 clone 53-6.7, Gr1 clone RB6-8c5, and CD11b clone M1/70 antibodies from Biolegend. Dead cells were excluded via 7AAD negativity. Data was acquired on a FACSCanto using FACSDiva software (BD Biosciences) and analyzed on FlowJo software vX10.07r2.

Tumors were harvested on day 5 and homogenized in RPMI using a gentleMACS Octo Dissociator (Miltenyi Biotec) on m_lung_01_01 and m_lung_02_01settings. Cells were separated with Lymphoprep (Stem Cell Technologies) according to the manufacturer’s instructions and stained for sorting with Zombie UV Fixable Viability Dye (BioLegend) as well as antibodies specific for CD45.2 (clone 104; Biolegend), CD11b (clone M1/70; Biolegend), and Gr-1 (clone RB6-8C5; Biolegend), CD4 (clone RM4-5; Biolegend), CD19 (clone 1D3; eBiosciences), and NK1.1 (clone PK136; Biolegend). Representative FACS plots from sorting are shown in Supplemental Figure 4.