Zen lsm imaging software

To visualize binding of CRT-targeting L-ASNases to IR-treated tumor cells, CT-26 and MC-38 cells (1 × 10⁴) were cultured on chamber slides (Thermo Scientific, USA) for 48 h and then treated with 10 Gy IR. At 48 h post-IR, the cells were fixed for 15 min with 1% PFA and washed with PBS containing 1% FBS. Then, the cells were stained with 100 nM CRT-targeting L-ASNases for 1 h on ice. After washing thoroughly with PBS containing 1% FBS, the cells were incubated for 1 h with anti-His tag monoclonal antibody (1:1000 dilution), followed by Alexa Fluor 488-conjugated secondary antibody (5 μg/mL) for 1 h. Cell membranes were stained with wheat AF555-conjugated wheat germ agglutinin (WGA) (1:5000 dilution). Finally, the cells were mounted in DAPI solution (Thermo Fisher Scientific, USA) to stain the nuclei. Immunofluorescence signals were detected using a LSM510 confocal microscope (ZEISS, Germany) and analyzed by ZEN-LSM imaging software (ZEISS, Germany).

The cultured cancer cells were grown on a glass coverslip for 48 h and treated with anticancer agents (15 µM GEM, 3 µM MTX, or 25 µM DOX) for 4 h [8 (link)]. The cells were fixed with cold acetone for 10 min and incubated with 1% bovine serum albumin (BSA) in PBS for 10 min. Then, the cells were incubated with a mouse anti-CRT antibody (1:1000 dilution; Abcam) for 2 h on ice. After washing with PBS five times, the cells were incubated with an Alexa Fluor 488-conjugated anti-mouse IgG (H + L) Cross-Adsorbed antibody (1:2000 dilution; Thermo Fisher Scientific) for 1 h on ice and washed five times. To stain cell membranes, the cells were also incubated with Alexa Fluor 555-conjugated wheat germ agglutinin (WGA) (1:5000 dilution; Thermo Fisher Scientific). Finally, the cells were mounted using DAPI anti-fade mounting solution (Thermo Fisher Scientific). The fluorescence signals were imaged using an LSM510 confocal microscope and analyzed with ZEN-LSM imaging software (ZEISS, Jena, Germany).