Alexa fluor plus 488 conjugated goat anti rabbit antibody

Purified NK cells and Raji cells were mixed with PBS or 161519 TriKE (10 μg/mL) and incubated for 30 min at 37 °C. The cells were then fixed in 1% paraformaldehyde and mounted on slides. Samples were blocked for 1 h at room temperature in 1% bovine serum albumin/PBS and stained with CD56 antibody (3576S; Cell Signaling Technology, Danvers, MA, USA) and CD19 antibody (ab134114; Abcam, Cambridge, UK) followed by Alexa Fluor Plus 647-conjugated goat anti-mouse antibody (A32728; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor Plus 488-conjugated goat anti-rabbit antibody (A32731; Thermo Fisher Scientific). All slides were stained with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific) for 4 min and mounted on coverslips in ProLong™ Gold anti-fade solution (Thermo Fisher Scientific). All procedures were conducted at room temperature. Slides were visualized using a LSM880 confocal laser scanning microscope (Zeiss, Oberkochen, Germany).

After treatment with oxaliplatin (10μM or 100 μM) or cisplatin (100 μM) for 4 h, CT26 cells were collected, washed twice with PBS and fixed in 0.25% paraformaldehyde in PBS for 5 min. After washing again twice in cold PBS, cells were incubated with the anti-calreticulin antibody (Abcam, ab2907) for 30 min at 4 °C, diluted in cold blocking buffer (2% fetal bovine serum in PBS), followed by washing and incubation with Alexa Fluor Plus 488-conjugated goat anti-rabbit antibody (A32731; Thermo Fisher Scientific) for 30 min at 4 °C. Each sample was then analyzed by flow cytometry on a FACS Celesta flow cytometer (BD Biosciences). Isotype-matched IgG antibodies were used as a control. The data were analyzed using FlowJo software (Tree Star).

After treatment with oxaliplatin (10μM or 100 μM) or cisplatin (100 μM) for 4 h, CT26 cells were placed on ice, washed twice with PBS and fixed in 0.25% paraformaldehyde in PBS for 5 min. Then the cells were washed twice in PBS, and stained with primary antibodies against CALR (Abcam, ab2907) for 30 min at 4 °C. After three washes in cold PBS, the cells were incubated for 30 min with Alexa Fluor Plus 488-conjugated goat anti-rabbit antibody (A32731; Thermo Fisher Scientific). Subsequently, the cells were fixed with 4% paraformaldehyde for 20 min. All slides were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) for 4 min and mounted on coverslips in ProLong™ Gold antifade solution (Thermo Fisher Scientific). Slides were visualized using an LSM880 confocal laser scanning microscope (Zeiss, Oberkochen, Germany).