Cytoperm cytowash

Cells were dissociated into single cells using TrypLE (Life Technologies) for 5 min at 37°C, pelleted, and fixed in Cytofix/cytoperm (BD Biosciences) for 10 min at 4°C. Cells were then incubated with 1:100 anti-cTnT antibody (Thermo Scientific MS-295-P), 1:100 CD31 (Dako M082329-2), FSP-1 (Millipore 07-2274), alpha smooth muscle actin (Abcam ab32575), or their isotype control, followed by incubation with secondary antibodies conjugated with Alexa 647 (Life Technologies) (Table S3). Every incubation step was performed in cytoperm/cytowash (BD Biosciences). Samples were run on a BD flow cytometer and analyzed using the FlowJo software.

Cells were differentiated in a tissue culture plate as described earlier. Without being lifted from the plate, the differentiated cells were incubated in the presence or absence of LPS for 20 hours. For on-plate imaging, U937 cells were differentiated and cultured in a Nunc Lab-Tek II chamber slide. Brefeldin A (Biolegend) was added to cells either simultaneously with LPS or 4 hours after the LPS was added, as indicated in the legends. After incubation, the cells were treated with trypsin (for flow cytometry analysis only), fixed with cytofix/cytoperm (BD Biosciences), washed with cytoperm/cytowash (BD Biosciences), and incubated with primary antibodies (BD Bioscience) according to the manufacturer’s instructions. All of the primary antibodies used for intracellular staining were the corresponding capture antibodies used in the SCBC (table S3). The cells were then stained with Alexa Fluor 488–conjugated goat anti-rat antibodies specific for IL-6, IL-10, or GM-CSF (Invitrogen) or with Alexa Fluor 647–conjugated goat anti-mouse antibodies specific for IL-1β, TNF-α, MIP-1β, or IL-8 (Invitrogen). Stained cells were analyzed with a BD Accuri C6 flow cytometer. On-chip staining was imaged with an EVOS FL Auto (AMAFD1000) microscope.