Annexin 5 allophycocyanin

Cellular apoptosis was analyzed using the FACScan instrument (BD Biosciences, San Jose, CA, USA). The cells were harvested and washed in phosphate-buffered saline, then subsequently stained with annexin V-allophycocyanin and propidium iodide 7-amino actinomycin D (BD Pharmingen, San Diego, CA, USA). Cells were quantified using a BD FACSVerse flow cytometer (BD Biosciences). Quantification of the apoptotic fraction included early and late apoptotic cells. All data were analyzed using Kaluza software 1.1 (Beckman Coulter, Brea, CA, USA). Representative data for three independent experiments are presented.

HS201, a novel photosensitizer made of VP (Novartis Pharmaceuticals, Basel, Switzerland) tethered to an Hsp90 small molecule inhibitor (HS10), was used for in vitro and in vivo imaging and PDT. HS201 was developed and supplied by Haystead Lab (Department of Pharmacology and Cancer Biology, Duke University) as previously described.23 (link) Anti-PD-L1 antibody (clone; 10F.9G2) was purchased from Bio X cell (New Hampshire USA) and used for animal treatment. Annexin V-Allophycocyanin was purchased from BD Biosciences (California USA) and 7-AAD from Beckman Coulter Inc. (California, USA). Detailed information of antibodies used for flow cytometry is shown in online supplemental methods.

Surface expression of Ro60 was assessed on nonfixed, nonpermeabilized, early apoptotic fibroblasts gated as annexin V+, PI−. Cells (apoptotic) were incubated with 300 μg/mL CHB IgG (isolated from an anti-SSA/Ro60 and Ro52-SSB/La-positive mother of a child with CHB [no antibodies to dsDNA]) or CHB IgG preincubated with peptide (30 minutes prior to addition to cells) for 45 minutes at room temperature. Control IgG obtained from a pool of healthy donors (300 μg/mL) that was negative for anti-SSA/Ro and SSB/La was also used. After incubation, cells were washed twice (phosphate-buffered saline/1% bovine serum albumin/0.02% sodium azide) and stained with anti-human IgG–fluorescein isothiocyanate (1:200) for 30 minutes. For apoptotic cells, 5 mL annexin V-allophycocyanin (BD Biosciences) and 5 mg/mL propidium iodide (Invitrogen) in annexin V binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 2.5 mmol/L CaCl2 [pH 7.4]) were added for 15 minutes at room temperature. Binding was assessed on a LSRII flow cytometer (BD Biosciences). The binding of CHB IgG to apoptotic cells is reflected by the percent of positive cells (after staining with anti-IgG fluorescein isothiocyanate and gating events versus control IgG).

Cells were washed twice with cold PBS, and then resuspended in 1× binding buffer (BD Biosciences, San Jose, CA, USA) at a concentration of 1×10⁶ cells/mL. Then, 100 mL of the solution (1×10⁵ cells) was transferred to a 5 mL culture tube and stained with 5 mL each of allophycocyanin–annexin V (BD Biosciences) and 50 mg/mL propidium iodide (Thermo Fisher Scientific). The cells were gently mixed and incubated at room temperature for 15 minutes. For assessment of apoptosis, 400 mL of 1× binding buffer was then added to each tube and the samples were analyzed by flow cytometry using a LSRII instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
The induction of apoptosis was also monitored by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The TUNEL assay was performed according to the guidelines recommended by the TUNEL Apoptosis Kit (R&D Systems, Inc.).