Alexa fluor 647 conjugated annexin 5

Alexa Fluor 647-conjugated Annexin-V (Invitrogen) was used to perform the apoptotic cell detection assays following the manufacturer’s instructions. Cells were treated for 24 h, acquired in FACSCanto II Cytometer (BD Biosciences), and analysed using FlowJo software v9.3.

Nε-acetyl-lysine (AcK) was purchased from Chem-Impex International (Wood Dale, IL). Diaphorase (#D5540), resazurin (#199303), NAD⁺ (#N7004), NADP⁺ (#N5755), G6P (#G7879), DSS (#S1885), CHX (#C7698), SAHA (#SML0061), NAM (#72340), MG132 (#M7449), phenylmethanesulfonyl fluoride (#P7626), sodium fluoride (#S1504), sodium orthovanadate (#S6508), ATP (#A2383), and 1 M manganese(II) chloride solution (#M1787) were obtained from Sigma-Aldrich. Sequencing grade chymotrypsin (#11418467001) and DNase I (#10104159001) were purchased from Roche Diagnostics (Mannheim, Germany). InstantBlue Coomassie Protein Stain (#ab119211) was purchased from Abcam (Cambridge, UK). Protein quantification was performed with QPRO-BCA Kit Standard from Cyanagen (Bologna, Italy). HisPur Ni-NTA Resin (#TS-88222) was purchased from Thermo Scientific (Waltham, MA). Alexa Fluor 647-conjugated Annexin V (#A23204) was purchased from Invitrogen (Waltham, MA). Aprotinin (#616370), Leupeptin (#108975), Pepstatin A (#516481), and NADPH (#481973) were obtained from EMD Millipore. DNA oligomers were obtained from Sigma-Aldrich unless otherwise stated. Enzymes and buffers for molecular biology were purchased from NEB (Ipswich, MA, USA).

FACS analysis was performed with a BD LSRII flow cytometer (BD Biosciences, UK). After 1 to 5 weeks of co-culture, nonadherent and adherent cells were harvested through trypsinization. Recovered cells were resuspended and stained in Annexin binding buffer (BD Biosciences) and with anti-SCA-1-PE or anti-CD56-PE or anti-CD31-PE, and anti-CD45-APC-Cy7, anti-CD34-Percp, anti-CD38-PE-Cy7 antibodies, and Lin-FITC (BD Biosciences), as well as with AlexaFluor647-conjugated Annexin-V (Invitrogen) and DAPI (Sigma). Only viable (both DAPI and Annexin-V negative fraction) human hematopoietic cells (CD45-APC-Cy7 positive and Sca-1-PE or CD56-PE or CD31-PE negative) were assessed for all analyses. Cell viability was expressed as the percentage of DAPI- and Annexin-V-negative cells within human hematopoietic cells.

Active caspase 3 was detected using CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (eBioscience) according to the manufacturer’s instructions. Cells were incubated with FITC-DEVD-FMK or Z-VAD-FMK for 30 min at 37°C prior to surface staining. The cells were then fixed with 0.8% paraformaldehyde in PBS followed by FACS analysis. For the detection of Annexin V, cells were stained with surface markers, washed with binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂ in PBS), and incubated with Alexa Fluor 647-conjugated Annexin V (Invitrogen) for 10 min at room temperature. After two washes with binding buffer, cells were fixed with 0.8% paraformaldehyde in PBS and subjected to FACS analysis.

Aliquots of 5 × 10⁵ cells were centrifuged at 520 × g and rinsed twice with Dulbecco's Phosphate Buffered Saline (DPBS) before resuspension in Annexin V binding buffer (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl₂, pH 7.4). Cells were labeled with Alexa Fluor 647-conjugated Annexin V as per manufacturer's instructions (Thermo Fisher scientific) and propidium iodide (PI; 4 ng/mL) in the dark, on ice, for 30 min prior to analysis.

Treated or control THP-1 or JY cells seeded into 24-well plates were labeled with Sytox Green Dead Cell Stain (Thermo Fisher Scientific) and AlexaFluor647-conjugated annexin V (Thermo Fisher Scientific) at dilutions of 1:1,000 and 1:20, respectively, in annexin binding buffer for 15 min at room temperature. Fluorescence intensities of individual cells were subsequently measured using a NovoCyte 3000RYB flow cytometer (ACEA Biosciences, San Diego, CA, United States). Sytox Green and AlexaFluor647 fluorophores were excited at 488 and 640 nm, respectively, and emitted intensities were measured using 530/30 and 660/20 emission filters, respectively. During data analysis, the fraction of Sytox Green and annexin V negative living cells was calculated for each sample using FCS Express.

Early apoptotic states of transduced T cells were evaluated using the Alexa Fluor 647 conjugated Annexin V (Thermo Fisher Scientific). In brief, a total of 1 × 10⁶ cells were incubated in 100 μL of annexin‐binding buffer (10 mm 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid, 140 mm NaCl and 2.5 mm CaCl₂, pH 7.4) and stained with 2 μL of the dye. The cells were diluted in 400 μL of annexin‐binding buffer and analyzed on BD LSR Fortessa after 15 min incubation at room temperature. Cells negative for Annexin V Alexa Fluor 647 nm and 4,6‐diamidino‐2‐phenylindole were considered viable cells. Data were analyzed using FlowJo software.