Cell dissociation buffer

The SW480, SW620, HT29, and HCT116 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM/F‐12, Gibco). The HL60 cell line was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Thermo Fischer Scientific). Media were supplemented with 10% fetal bovine serum (Sigma), 2 mM Glutamax (Thermo Fisher Scientific), and penicillin 100 units/ml streptomycin 100 μg/ml (Sigma). Phase contrast images of the SW620 and SW480 cell lines grown in flasks showed a more epithelial‐like morphology for SW480's and a more fibroblast‐like morphology for SW620's as shown in Figure S2. Cells were not “synced” to allow the natural cell cycle within sample variability of the cell lines. All experiments were done with passage numbers below 50. SW480, SW620, HT29, and HCT116 were washed with Dulbecco's phosphate buffered saline (DPBS) and gently retrieved from six‐well plates by incubating with cell dissociation buffer (Thermo Fisher Scientific) for 30 min, followed by centrifugation (100 Gs; 1 min) and resuspension in cell dissociation buffer. HL60 cells were retrieved from media by centrifugation (100 Gs; 1 min) and washed with DPBS once before resuspending in DPBS. When pipetted into the setup, cells sedimented onto the coverslip and showed no visible Brownian motion, remaining in a spherical shape.

RAW264.7 cells expressing green fluorescent protein (GFP) or GFP-PE_PGRS30 were collected 24 h post-transfection using Cell Dissociation Buffer (Gibco, United States) and stained with annexin V-PE (559763, BD biosciences) and 7-amino-actinomycin D (AAD, BD Biosciences) according to the manufacturer’s instructions. BAL cells treated with recombinant proteins for 24 h were collected using Cell Dissociation Buffer (Gibco) and stained with antibodies; PE-Cy7-conjugated anti-CD11c (N418, Biolegend Inc., San Diego, CA, United States), APC-conjugated anti-Siglec-F (E50-2440, BD Biosciences) and APC-Cy7-conjugated anti-CD45 (30-F11, Biolegend) antibodies for 20 min, and then stained with Annexin V-FITC (556570, BD Biosciences) and 7-AAD according to the manufacturer’s instructions. Flow cytometry data were collected using a FACSCanto II (BD Biosciences) instrument and analyzed using FlowJo (Tree Star, Inc., Ashland, OR, United States). Alveolar macrophages were defined as CD45-Siglec-F-and CD11c-positive cells (Misharin et al., 2013 (link)). The number of annexin V-positive cells was defined as the sum of the numbers of annexin V⁺/7-AAD⁺ cells and annexin V⁺/7-AAD-cells.

Bone marrow was flushed from mouse femurs and tibias with Ca²⁺ and Mg²⁺-free Hepes-buffered saline solution (HBSS). Pellets were stored at −80°C for protein analysis by western blot, or were frozen in liquid nitrogen in 10% DMSO/90% FCS for cell culture experiments. For in vitro differentiation of bone marrow-derived macrophages, the method was adapted from (39 (link)). Cells were plated at 2 × 10⁶ cells per 10 cm dish in 10 ml macrophage media consisting of αMEM (Gibco) supplemented with 10% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine and 10 ng/ml M-CSF (Peprotech). Functional assays were performed starting on day 7 of culture. For polarization, macrophages were removed from plates using Cell Dissociation Buffer (Gibco), washed, and plated in either M0 conditions (macrophage media), M1-polarizing conditions (macrophage media supplemented with 100 ng/ml LPS and 20 ng/ml recombinant IFNγ) or M2-polarizing conditions (macrophage media supplemented with 20 ng/ml recombinant murine IL-4) for 24 h before analysis. To measure CD206 expression on M2 polarized mouse macrophages, cells were removed from plates using Cell Dissociation Buffer (Gibco) and stained for subsequent analysis by flow cytometry as outlined below. To measure cytokine production by M1 polarized macrophages, supernatants were harvested and analyzed by multiplexed ELISA.