Alexa fluor 488 anti hsp70 antibody

A FOSCAN-mediated PDT was performed as described in paragraph 2.6. Next, 2 h after PDT, cells were collected for cell surface CRT and HSP70 expression by staining with recombinant Alexa Fluor^® 405 anti-calreticulin antibody (Clone EPR3924, Abcam, Cam- bridge, UK) and Alexa Fluor^® 488 anti-HSP70 antibody (Clone W27, Biolegend, San Diego, CA, USA) on an LSR-II flow cytometer. HMGB1 expression was explored by staining with anti-HMGB1/HMG-1 antibody (Novus Biologicals, Littleton, CO, USA) on ice for 30 min, washed three times, and stained with recombinant goat anti-rabbit IgG (H + L) secondary antibody, Alexa 568-PE (Invitrogen, Waltham, MA, USA) before analyzing on an LSR-II flow cytometer.

The 4T1-luc2 tumor cells were treated with crassolide (50 to 3.125 µM) for 24 h. Aliquots of 1 × 10⁶ 4T1-luc2 tumor cells were harvested, washed with PBS, and stained with PE anti-HSP90 antibody (LSBio, LS-63265), Alexa Fluor 488 anti-Hsp70 antibody (BioLegend, #648004), PE anti-HMGB1 antibody (BioLegend, #651404), PE anti-calreticulin (Cell signaling, #62304), APC anti-PD-L1 (BioLegend, #124312), Alexa Fluor 488 anti-CD24 (BioLegend, #101816) in 0.5% bovine serum albumin (BSA, Bionovas) in PBS, and then analyzed using flow cytometry (BD FACSVerse, San Jose, CA, USA).