Ab2907

BHK-21 cells were seeded in ibidi slides (15 u-slide angiogenesis, ibidi) and infected with DENV-2 (TSV01) at MOI 1. 48 h post-infection, the cells were fixed with 4% PFA for 20 min at room temperature (RT), permeabilized with 1× PBS + 0.05%Triton X-100 for 15 min at RT and blocked with 1× PBS, 1% BSA for 1h at RT. Cells were then incubated with monoclonal Abs SIgN-3C (2 ug/ml) or rabbit anti-calreticulin (Abcam Ab2907) (1:100) or rabbit anti-giantin (Abcam Ab37266) (1:1000) for 1 h at RT. Secondary Abs goat anti-rabbit AF568 and goat anti-human AF488 (MolecularProbes) were used at 1:2000 dilution. Hoechst 33342 (Molecular Probes) was added for 5 min at RT. All washes were performed with PBS for 10 min at RT. Antibody binding was visualized with an Olympus confocal microscope at 100X magnification, using oil.

Human osteosarcoma U2OS cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES in a humidified atmosphere containing 5% CO₂ at 37 °C. Cell culture media and supplements were from Gibco-Invitrogen (Carlsbad, CA, USA), and all plastic supplies from Corning (Corning, NY, USA).
Geneticin (Neo) and hygromycin (Hygro) were purchased from Invivogen (San Diego, CA, USA). Biotin and avidin were purchased from Sigma-Aldrich (St. Louis, MI, USA). Rapamycin and bafilomycin A1 were purchased from LC Laboratories (Woburn, MA, USA).
Primary antibodies used in this study are: rabbit anti-GAPDH (#2118, Cell Signaling Technology), rabbit anti-B4GALT1 (PAB20512, Abnova), rabbit anti-CALR (ab2907, Abcam), mouse anti-streptavidin (sc-52234, Santa Cruz), mouse anti-SQSTM1 (sc-28359, Santa Cruz). Secondary antibodies used in this study are from Southern Biotech (HRP-conjugated; Birmingham, AL, USA) or Thermo Fisher Scientific-Molecular Probes (Alexa 488-, Alexa 546- or Alexa 647-conjugated; Waltham, MA, USA).

This procedure was based on a published method (Liu et al., 2020 (link)), but was performed with some modifications. Briefly, HSC-3 and UT-SCC-24A cells were seeded, 300,000 cells/well, in 6-well plates and left to adhere overnight. The media was then removed and replaced by 2 ml complete DMEM/F-12 containing 5, 7.5, 10 or 20 µM of MPM-2:0, MPM-6:0, MPM-3:2 or MPM-4:2, or 50 µM cisplatin. Some cells were left untreated. The cells were then incubated for 24 h. Next, the cell supernatants were collected before the cells were trypsinized and spun down with the supernatants. The cells were then washed in PBS before being incubated with the viability dye Zombie Violet (#423114, BioLegend, San Diego, CA, United States) diluted 1:500 in PBS for 20 min. Next, the cells were incubated with an anti-calreticulin antibody (#ab2907, Abcam) diluted 1:100 in FACS buffer (2%BSA in PBS) for 30 min. The cells were then fixed in 4% formaldehyde for 15 min and washed twice in FACS buffer before being incubated with the secondary antibody (#A11034, Thermo Fisher Scientific) diluted 1:250 in FACS buffer for 30 min. The cells were washed in FACS buffer and analyzed on a LSR Fortessa (BD Biosciences) within 1 week. Analyses were performed in FlowJo™ v.10 (https://www.flowjo.com/).

Cell death from CP1 or MG1655 (MOI 1 or 10) and cancer cell co-culture was measured by supernatant lactate dehydrogenase (LDH; Cytotoxic 96 Non-Radioactive Cytotoxicity Assay, Promega). For in vitro ICD assays, 1 μM mitoxantrone was used as a positive control. Supernatants were collected and cell counts performed after 72 h for quantifying secreted ATP (Bioluminescent Assay Kit, Sigma) and high mobility group protein B1 (HMGB1; ELISA, Tecan Trading). Also after 24 or 72 h, cells were incubated with rabbit anti-calreticulin (Abcam ab2907 1:1000) for 60 min, followed by Alexa Fluor 488 anti-rabbit secondary (Invitrogen A11008 1 μg/ml) for 30 min, and analyzed by flow cytometry. For in vivo ICD assays, HMGB1 immunofluorescence (IF) was quantified as the percentage of HMGB1⁻ nuclei and calreticulin IF was analyzed for cell surface staining. In vitro, caspase 3/7 activity was assessed at 6 or 24 h (Caspase-Glo 3/7 Assay, Promega). Early (Annexin V⁺ PI⁻) and late (Annexin V⁺ PI⁺) stage apoptosis were analyzed at 24 h by flow cytometry (Annexin V Apoptosis Detection Kit, eBioscience). Phosphorylated MLKL, MLKL, RIP1, and full length and cleaved PARP were analyzed by Western blot. As indicated, select experiments included the addition of 50 μg/ml gentamicin 2 h after co-culture initiation.

Cells were collected, washed twice with 1x PBS and fixed in 0.25% paraformaldehyde in 1x PBS at 4 °C. After 5 min incubation, cells we washed twice in cold 1× PBS and incubated for 30 min at RT with anti-calreticulin (CRT; Ab2907, Abcam, Cambridge, UK) or ERp57 (Ab10287, Abcam) primary antibody diluted (1:50) in cold blocking buffer (2% FCS in 1× PBS) and then incubated for 30 min with an Alexa488-conjugated monoclonal secondary antibody (A11034) diluted (1:50) in blocking buffer. Isotype-matched Alexa488-conjugated IgG antibodies were used as a control. Samples were then analyzed by flow cytometry. Data were recorded statistically (10,000 events/sample) using the CellQuest Pro software and analyzed using the Flow-Jo 8.8.7 software Results were expressed as mean fluorescence intensity (MFI). Samples were also analyzed by fluorescence microscopy (Nikon Eclipse Ti-U, Nikon Instruments Korea, South Korea).

For histopathological analysis of various proteins, the formaldehyde-fixed paraffin sections of tissue were incubated with primary antibodies, anti α smooth muscle actin (α-SMA) (ab-7817; 1:200; Abcam), cyclophilin A (ab41684; 1:200; Abcam), calreticulin (ab-2907; 1:100; Abcam) and CD 47 (B6H12.2; 1:100; Novus Biologicals) overnight at 4 °C. Species- and isotype-matched IgG was used as negative control. Anti-rabbit IgG-HRP (ab-97051) and anti-mouse IgG HRP (ab-6789) were used as secondary antibodies at a dilution of 1: 400 and 1:200, respectively. Quantitative analysis was performed manually by analyzing diaminobenzidine-tetrahydrochloride- (DAB) positive areas using ImageJ software.