Annexin 5 binding buffer

Cells were plated at 5 × 10⁴ cells/cm² and allowed to attach for 3 hours. Medium was exchanged to medium supplemented with vehicle or 200 μM 1-EBIO and cells were incubated for 24 h. To quantify the percentage of apoptotic and dead cells, cells were trypsinized, washed in PBS, and incubated in 0.1% Live/Dead Fixable Violet Dead Cell stain for 30 min. Cells were washed in Annexin V Binding Buffer and incubated in Annexin V conjugated to FITC diluted 1:125 in Annexin V Binding Buffer (Invitrogen). For cell cycle analysis, cells were fixed in 70% ethanol and washed in phosphate-buffered saline (PBS). Cells were incubated in 50 ng/mL propidium iodide (Invitrogen), 250 ng/mL RNase diluted in PBS for 1hr. Cell fluorescence was detected using an LSR II (Becton Dickenson) fluorescent cell analyzer and data were analyzed with FlowJo software.

Cortical organoids, primary fetal neurons, and astrocytes were manually dissociated and resuspended in Annexin V binding buffer (Invitrogen). Next, Annexin V-CF488A conjugate (Biotium, Inc., Hayward, CA, USA) was added, followed by Hoechst 33342 (Chemometec). After a PBS wash, the cells were resuspended in Annexin V binding buffer (Invitrogen) containing 10 μg/mL propidium iodide (Chemometec). Cells were loaded into NC‐Slide A2 chambers, and the “Annexin V Assay” was run with the NucleoCounter NC-3000.

One million PBMC were stimulated with TLR-L and naltrexone for 24 h before being resuspended in 1× annexin V binding buffer (eBioscience) and incubated with 5 µl annexin V APC (eBioscience) for 20 min. Cells were then washed in 1 ml 1× annexin V binding buffer and resuspended in 200 µl 1× annexin V binding buffer. 5 µl 7AAD was then added, and data were collected using the BD Canto. Data were analyzed using FlowJo software.

Cell viability and cell death were determined using commercially available kits (PrestoBlue^® cell viability reagent, Invitrogen^TM; Cytotoxicity Detection Kit (LDH), Roche), following the provider’s instructions. Briefly, 10,000 cells were seeded in a 96-well plate and incubated overnight. Culture media was transferred into a new 96 well plate for the LDH assay. Cells left in the original plate received fresh media containing PrestoBlue^®. The fluorescence intensity (PrestoBlue^® assay) was measured after 1 h; the absorption (LDH assay) was measured according to manufacturer’s instructions, using the plate reader MWGt Synergy HT (BioTek Instruments) and the software Gen5 2.01 (BioTek).
In addition, cell viability and cell death were measured using flow cytometry. For this, cells were grown in 75 ml culture flasks and harvested by gentle trypsinization (0.25% Trypsin-EDTA; Gibco^®, Thermo Fisher Scientific). Cells were centrifuged at 400 × g for 5 min and washed twice with 1× Annexin V Binding Buffer (eBioscience). Cells were labelled with Alexa Fluor® 647 Annexin V (Biolegend) and 7-Amino-Actinomycin (7′AAD) (BD Pharmingen). Data was recorded by flow cytometry to determine the number of Annexin V/7′AAD-positive cells.