Ack lysis solution

To prepare single cell splenocyte suspensions, spleens were mechanically dissociated through 70µm cell strainers, red blood cells were lysed with ACK lysis solution (Gibco), then washed with RPMI supplemented with 10%FBS/1X Pen/Strep and L-Glutamine (cRPMI). Live splenocytes were quantified using a Cellometer Auto 2000 and AOPI live/dead dye (Nexcelom), then adjusted to a final concentration of 1x10⁷ cells/mL in cRPMI. For each sample, 1x10⁶ live splenocytes were restimulated for 96 hours with 100 µg/mL recombinant Tc24-C4 protein or cRPMI (unstimulated) at 37°C, 5% CO_2. As a positive control, splenocytes incubated for 6 hours with 20ng/mL phorbol myristate acetate (PMA) (Sigma-Aldrich)/1mg/mL Ionomycin (Sigma-Aldrich) were included.

Single cell suspension from mouse lungs was prepared as previously described [31 (link)]. In all experiments, left lungs were aseptically removed, minced using sterile razor blades (Fisher Scientific), and incubated in RPMI 1640 containing 1 mg/ml collagenase (Sigma) and 30 ug/ml DNase (Sigma) at 37°C for 60 min. To achieve a single-cell suspension, lung fragments were pressed through a 70 μm pore nylon cell-strainer (BD Falcon) using the flat-end of a sterile 3 ml syringe plunger. Cells were washed twice in complete RPMI (RPMI supplemented with 2mM L-glutamine, 25mM HEPES, 10% fetal bovine serum and 55 μM 2-mercaptoethanol) and red blood cells were lysed by incubation in ACK lysis solution (Thermofisher Scientific; 0.156 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.2) for 5 min at room temperature. Cells were then washed again in complete RPMI and counted, using trypan blue to exclude dead cells. Total number of cells from the lungs was enumerated using a hemocytometer in conjunction with light microscopy [31 (link)].

Bone marrow‐derived macrophages (BMDM) were prepared essentially as described before (Marim et al, 2010). Briefly, mice femurs and tibias were dissected and sterilized with 70% ethanol. The bones were flushed with a syringe filled with advanced RPMI 1640 (Life Technologies) to extrude bone marrow. Cell suspensions were filtered through a 100‐μm cell strainer and incubated for 2 min in ACK lysis solution (Thermo Fisher Scientific) to lyse red blood cells. The bone marrow cells were cultured and differentiated in advanced RPMI 1640 supplemented with 2 mM l‐glutamine, 10% (v/v) FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 ng/ml murine M‐CSF (R&D System) using non‐tissue culture treated Petri dishes (BD Biosciences). Three days after seeding, fresh murine M‐CSF (final concentration of M‐CSF 50 ng/ml) was added and media was changed at day 5 of culture. BMDM were used for experiments at day 7 in vitro. Immunostaining with CD68 (Figs 3C and 4C) as well as flow cytometry (see below) using anti‐CD11b antibody (Fig EV1A) was used to confirm the identity of these cells.

Mice “at rest” were culled and blood extracted from the vena cava, followed by perfusion using 20 mL of PBS (Sigma) containing 1mM EDTA (Sigma) before analysis of the cellular content of a number of tissues.
Dissected spleens were crushed onto 70 μm nylon mesh filters and washed with PBS (Sigma). Spleen and blood cell suspensions then underwent red blood cell lysis (ACK lysis solution, Thermo Fisher Scientific) before washing, ready for cellular content analysis. The serum from centrifuged blood was aspirated and taken for multiplex analysis as detailed below.
Shaved lower dorsal skin was dissected and chopped into fine pieces, followed by digestion in 4ml of digest cocktail (Hanks buffered saline solution (HBSS) containing collagenase D (1mg/mL Roche), dispase II (500 μg /mL, Roche) and DNase I (100 μg/mL, Roche)) for 1.5 hours at 37°C with shaking. Perfused lungs were dissected and chopped into fine pieces before digestion in 5ml of digest cocktail (RPMI containing DNase I (100 μg/mL, Roche), dispase II (800 μg /mL, Roche) and collagenase P (200 μg/mL, Roche)) at 37°C for 1.5 hours, with inversion after 45 min. Enzymes were neutralized by adding 20 μL of fetal bovine serum (FBS) to each tube before skin or lung cell suspensions were filtered through 40 or 70 μm nylon mesh filters, respectively, and washed for cellular content analysis.

Blood samples were taken from tail tips of resting WT or iCcr heterozygous animals. After red blood cell lysis (ACK lysis solution, Thermo Fisher Scientific), whole RNA was extracted using the RNeasy mini kit with DNase treatment (QIAGEN). RNA was then reverse transcribed into cDNA using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and cDNA was used in the analysis of Ccr1, 2, 3, 5 and Cxcr2 expression by QPCR (PerfeCTa® SYBR® Green FastMix, Quanta Biosciences). All QPCRs were performed in a Prism 7900HT Fast Real-Time PCR system (Applied Biosystems). iCcr expression was calculated using standard curves specific for each gene and results were normalized to the expression of the housekeeping gene GAPDH. QPCR and standard primers used in these analyses are detailed in Table S3.