Annexin 5 fitc pi kit

Cellular apoptosis was investigated using the FITC Annexin V/PI kit and following the recommended protocol (BioLegend Inc., San Diego, USA). Briefly, 1 × 10⁶ cells from different treatments were trypsinized, rinsed twice with cold cell staining Buffer, and suspended in 100 μl of binding buffer. Subsequently, 5 μl Annexin V-FITC was added and incubated for 10 min in dark condition at RT, followed by the addition of 10 μl PI for 5 min before flow cytometry (BD FACSVia Flow Cytometer, BD Biosciences, USA).

The apoptosis percentages of male germ cells of wild type and P63^(+/−) mice were detected using the Annexin V-FITC/PI kit by flow cytometry pursuant to the manufacturer’s instruction (Biolegend, London, UK). Male germ cells were obtained from P63^(+/−) mice and wild-type mice using two-step enzyme and followed by the differential plating and culture for 24 h. It was feasible to identify and quantify apoptotic cells by conjugating FITC to Annexin V using flow cytometry. The cells were simultaneously stained with Annexin V-FITC (green fluorescence) and the non-vital dye PI (red fluorescence), which allowed the discrimination of intact cells (FITC⁻PI⁻), early apoptotic (FITC⁺PI⁻) and late apoptotic or necrotic cells (FITC⁺PI⁺). The procedure was carried out according to the protocol as described previously^{77 (link)}.

Human Sertoli cells seeded in 12-well plates (2.5 × 10⁴ cells/well) were exposed to 100 ng/ml recombinant human BMP6 factor or 0.1% BSA (control) for 72 hours in DMEM/F-12 supplemented with 1% FBS, or BMP6 siRNA-1, or control siRNA for 48 hours as previously described. Cells were harvested and washed with cold PBS, and apoptosis percentages of human Sertoli cells were detected using the Annexin V-FITC/PI kit by flow cytometry pursuant to the manufacturer’s instruction (Biolegend, London, UK). It was feasible to identify and quantify apoptotic cells by conjugating FITC to Annexin V using flow cytometry. Staining cells simultaneously with Annexin V-FITC (green fluorescence) and the non-vital dye PI (red fluorescence) allowed the discrimination of intact cells (FITC⁻PI⁻), early apoptotic (FITC⁺PI⁻) and late apoptotic or necrotic cells (FITC⁺PI⁺).

The cells were treated with 0.175 μM, 575 μM, and 500 μM of PTXO, PINO, and LARI, respectively. After 24 hours and 48 hours of treatment, cells were harvested and stained using an annexin V-FITC/PI kit, and apoptosis was examined according to the manufacturer’s protocol (BioLegend, CA, USA). The cells were washed with phosphate-buffered saline (Gibco, Invitrogen, Carlsbad, CA, USA) and dissolved in 500 µl of 1×binding buffer containing 5 µl annexin and 10 µl PI. Then, it was incubated in the dark at room temperature for 15 min. Flow cytometry (BD FACSCalibur, BD Bioscience, CA, USA) was then used to assess the extent of annexin binding and PI staining.

C3G (purity ≥99%, Fig. 1), L-glutamic acid, Dulbecco’s modified Eagle medium (DMEM), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Antibodies against calpain, caspase12, CHOP, ERK, p-ERK (Thr202/Tyr204), β-actin and horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and Nrf2 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Bio basic (Toronto, Canada). Dimethylsulfoxide (DMSO) was purchased from Merck (Darmstadt, Germany). Penicillin-streptomycin solution was obtained from Corning Inc. (Corning, NY, USA). Annexin V FITC/PI kit was purchased from Biolegend (San Diego, CA, USA). The reagent kit for lactate dehydrogenase (LDH) was bought from Promega’s CytoTox 96™ (Madison, WI, USA) and 2′,7′- dichlorofluorescein diacetate (H₂DCF-DA) was obtained from Life technology (Carlsbad, CA, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA).
Fig. 1

Chemical structure of C3G

Poly(D,L-lactide-co-glycolide) (PLGA polymer with carboxylic terminal group, 50/50 of inherent viscosity midpoint 0.2 dL/g; M_W 10,000–18,000) was purchased from LACTEL Absorbance Polymers (Birmingham, AL, USA). D-mannitol and polyvinyl alcohol (PVA, MW 30,000–70,000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroform was purchased from Ruskhim (Moscow, Russia). Dimethyl sulphoxide (DMSO), Tween 80, and phosphate buffered saline (PBS) were purchased from Amreso (Solon, OH, USA). Trypsin and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). Ethylenediaminetetraacetic acid (EDTA), 2′-7′dichlorofluorescin diacetate (DCFH-DA), and paraformaldehyde were purchased from Serva (Heidelberg, Germany). DMEM and RPMI 1640 culture medium were purchased from Gibco (Waltham, MA, USA). 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT), giemsa, mowiol, and o-phthaldialdehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoSox Red was purchased from Invitrogen (Waltham, MA, USA). MitoStep was purchased from Immunostep (Salamanca, Spain). Annexin V-FITC/PI kit was purchased from Biolegend (San. Diego, CA, USA). TUNEL and Lipid Peroxidation (MDA) Colorimetric assay kits were purchased from Biovision (Milpitas, CA, USA). All other chemicals were used as HPLC grade or extra pure grade.