Anti crt antibody

1 × 10⁶ cells, rinsed and fixed with 2% w/v paraformaldehyde for 2 min, were washed three times with PBS and stained with an anti-CRT antibody (Affinity Bioreagents) for 1 h on ice. After washing, samples were incubated with an AlexaFluor 488-conjugated secondary antibody (Millipore) for 30 min and re-washed. Samples were analyzed with a Guava easyCyte flow cytometer (Millipore). For each analysis 10,000 events were collected. Control experiments included incubation with non immune isotypic antibody followed by the secondary antibody. The results were analyzed with the easyCyte software (Millipore).

To measure the levels of surface CRT, 1 × 10⁵ cells were washed with PBS, detached with Cell Dissociation Solution (Sigma-Merck), and incubated for 45 min at 4 °C with the anti-CRT antibody (Affinity Bioreagents; dilution 1/100), followed by an AlexaFluor488-conjugated secondary antibody (Abcam; dilution 1/50) for 30 min at 4 °C. After the fixation step in 2.5% v/v paraformaldehyde for 5 min at room temperature, samples were analyzed with a Guava EasyCyte (Millipore) flow cytometer equipped with the InCyte software (Millipore). Propidium iodide-negative, CRT-positive cells were counted. Cells incubated with an isotype control antibody, followed by secondary antibody, were included as control of specificity. The ATP release was measured on 100 μL of the cell culture medium with the ATP Bioluminescent Assay Kit (FL-AA, Sigma-Merck). The results were expressed as nmoles/mg cellular proteins. The release of HMGB1 was measured using the High Mobility Group Protein 1 ELISA kit (Cloud-Clone Corp., Houston, TX, USA), as per manufacturer’s instructions. The results were expressed in nmoles/mg cellular proteins.