Alexa fluor 647 conjugated anti crt antibody

To assess CRT expression by flow cytometry, 4T1 cells or EMT6 cells inoculated in six-well plates (5 × 10⁵ cells/well) were cultured with different concentrations of losartan and Dox-L for 48h. The treated cells were collected and sequentially incubated with a primary rabbit Alexa Fluor^® 647-conjugated anti-CRT antibody (dilution 1:50, Abcam, UK) for 20 min at room temperature. The cells were incubated in 500µL PBS containing 10% fetal bovine serum (FBS) and 1µg/mL DAPI (Biolegend, USA) before assessment with a flow cytometer (CytoFLEX LX, Beckman Coulter, USA). The mean fluorescence intensity of stained cells was gated on DAPI- cells.

Cell apoptosis was detected by Flow cytometry using FITC Annexin V Apoptosis Detection Kit I (556547, BD Pharmingen) according to the manufacturer's instructions. Cells (2×10⁵ cells/well in a 12-well plate) were treated with PBS, Mix, free cdG, or cdG/Mix for 24 hours. Cells were washed twice with PBS and then resuspended in 1× Binding Buffer. Cells were stained with Annexin V and PI for 15 mins at RT in the dark, and run on the CytoFLEX S flow cytometer (BECHMAN COULTER). Data from three independent experiments were analyzed using CytExpert software.
Calreticulin (CRT) expression was detected by Flow cytometry using Alexa Fluor 647-conjugated anti-CRT antibody (Ab196159, Abcam). Cells (2×10⁵ cells/well in a 12-well plate) were treated with PBS, Mix, free cdG, or cdG/Mix for 24 hours. cells were trypsinized, washed in cold PBS, and stained with anti-CRT antibody for 1 h at 4 °C, and stained with PI for 5 mins at RT in the dark then washed three times and re-suspended in cold PBS with 3% FBS and analyzed on a CytoFLEX S flow cytometer (BECHMAN COULTER). Data from three independent experiments were analyzed using CytExpert software.