Alexa fluor 647 conjugated donkey anti mouse igg

Immunofluorescence staining and flow cytometry were performed as previously described (18 (link)). Primary antibodies (table S5) were used at the following dilutions: rabbit anti-GFP (Invitrogen, A11122; 1:500), chicken anti-GFP (Abcam, ab13970; 1:1500), anti-αActinin (Sigma-Aldrich, A7811; 1:500), anti-Cx43 (Sigma-Aldrich, C6219; 1:200), anti-Lamp1 (Abcam, ab25245; 1:200), and anti–β-catenin (Abcam, ab16051; 1:200). Secondary antibodies including Alexa Fluor 488–conjugated donkey anti-rabbit IgG, Alexa Fluor 488–conjugated donkey anti-chicken IgG, Alexa Fluor 647–conjugated donkey anti-mouse IgG, cyanine Cy3-conjugated donkey anti-rabbit IgG, and cyanine Cy3-conjugated donkey anti-mouse IgG were all from Jackson ImmunoResearch Inc. Images were captured using EVOS FL Auto Cell Imaging System (Life Technologies). For quantification, 10 to 20 images were randomly taken under ×10 or ×20 magnifications at the same exposure setting and then counted in a double-blinded way. For flow cytometry, iCMs on d10 were harvested by trypsin digestion at 37°C for 5 min. Cells were fixed, permeabilized, probed for αMHC-GFP and cTNT, and then analyzed on a BD Accuri C6 or Cyan flow cytometer. FlowJo software (Tree Star) was used to analyze flow cytometry data.

Cell expression of the integrin subunits α₁ and α₂ was assessed by flow cytometry using the Agilent 2100 Bioanalyzer microfluidic system. Briefly, EOCs or HUVECs were resuspended at 200,000 cells per mL, placed on ice, and exposed to 2 μM Calcein AM (Life Technologies) in staining buffer (1% BSA in PBS) for 30 min. The cells were then pelleted by centrifugation and washed twice with staining buffer, after which they were resuspended in 100 μL staining buffer containing 10 μg/mL of appropriate primary antibody (integrin α₁: clone FB12, integrin α₂: clone P1E6; Millipore), or IgG control. After incubation for 30 min, the cells were centrifuged and the pellet was rinsed twice with 1 mL of staining buffer. Secondary antibody (Alexafluor 647 conjugated donkey anti-mouse IgG; Jackson Immunoresearch) was then applied at 10 μg/mL in staining buffer for 30 min. Finally, the cell pellet was rinsed twice with 1 mL staining buffer, resuspended at 1x10⁶ cells/mL in loading buffer, and then applied to the flow cytometry microfluidic chip (Agilent 2100 Bioanalyzer) according to the manufacturer’s instructions. For each antibody and cell type, flow assessment was run in triplicate. Flow cytometry for the β₁ integrin subunit was also conducted and is shown in Supplementary Figure 2.

Chemicals, reagents and plasmids were obtained from the following: Ficoll–Paque Premium (GE Healthcare), Clini-MACS CD3 reagents and CD14 microbeads (Miltenyi Biotech), Blebbistatin, ionomycin, apyrase and PMA (Sigma-Aldrich), brefeldin and monesin (Pharmingen), nonmuscle actin and ATP (Cytoskeleton), CellTracker Orange (Life Technologies), anti–UNC-45A (Enzo Life Sciences), anti-perforin, anti–CD3-PerCP, anti–CD56-allophycocyanin and anti–CD107a-PE (BD Biosciences), anti-NMIIA (Covance), anti–p-NMIIA H chain (Ser1943) (Cell Signaling), anti-NMIIA L chain (Ser20) (Abcam), anti-lysosomal associated membrane protein 1 (LAMP-1), anti-actin (Sigma), PE-conjugated anti-perforin (BD Pharmingen), mouse monoclonal anti-β actin for immunofluorescence (IF) (Sigma), Alexa Fluor 647–conjugated donkey anti-mouse IgG, Texas red goat anti-mouse IgG, Texas red-goat anti-rabbit IgG, FITC-donkey, and anti-mouse IgG (Jackson Immunoresearch Laboratories).