Annexin 5 apc

COS-7 cells were grown in six-well plates (Greiner) to 80% confluency, transfected the next day with plasmids encoding EGFP or EGFP-coupled RBD-probes and then cultured for additional 24 h in fresh culture medium. Cells were detached by trypsin/versene (Gibco®/Life Technologies, Darmstadt, Germany) and collected by centrifugation. The cell pellet was washed twice in 1 × PBS and re-suspended in 220 μl 1 × bindings buffer (BD Biosciences). The sample was divided in two: 100 μl sample were left untreated, the other 100 μl were supplemented with 2.5 μl Annexin V-APC (BD Biosciences). The different preparations were incubated for 5 min at 37°C and then for 25 min at room temperature in the dark. To determine the proportion of dead cells among the EGFP or EGFP-RBD-expressing COS-7 cells Annexin V-APC was measured using the FACS Calibur^R instrument (BD Biosciences) and plotted against EGFP. Subsequent propidium iodide (Merck Biosciences, Schwalbach, Germany) staining revealed that approximately 85% of the transfected, dead cells underwent apoptosis.

Proliferating podocytes were transfected with GFP-ev (empty vector), PACSIN2-wt, PACSIN2-S313E or PACSIN2-S313A using Lipofectamine 2000 [9 (link)]. After 72 h, cells were fixed with 4% PFA and stained with Annexin V-APC 1:50 (BD, Franklin Lakes, NJ, USA). The percentage of Annexin V-APC positive cells, in the GFP positive population, was measured by flow cytometry using BDaccuri (BD Life Sciences, Franklin Lakes, NJ, USA). A total of 10⁵ cells were detected in each sample.

After incubation of lymphocytes for 48 h at 37°C and 5% CO₂, the samples were centrifuged for 10 minutes at 300 G and 4°C, and then put on ice and decanted. Each pellet was resuspended in 200 μL lactated ringer's solution. A mix of monoclonal antibodies, Annexin V-APC (BD Biosciences, Heidelberg, Germany) and 7-Aminoactinomycin D (7AAD) (BD Biosciences, Heidelberg, Germany), was added, consisting of the following quantities: 20 μL of CD4-FITC, 20 of μL CD8-PE, 5 μL of Annexin V-APC, and 5 μL of 7-Aminoactinomycin. After 30 minutes on ice, another 200 μL of lactated ringer's solution (Braun, Melsungen, Germany) was added to each sample and lymphocytes were analyzed using a Gallios flow cytometer (Beckmann Coulter, Krefeld, Germany). In order to identify lymphocytes, forward scatter and sideward scatter were used. Lymphocyte subtypes were identified according to their CD4+ and CD8+ surface antigens. Apoptotic and necrotic rates were measured by Annexin V and 7AAD staining (Figures 1(a)–1(d)).

For cell cycle analysis experiments using propidium iodide (PI), cells were pelleted and fixed in 80% ice-cold ethanol and stored at 4°C until further processing. Cells were stained with PI at 50 μg/ml in PBS containing RNase A and analyzed by flow cytometry on the BD FACSCanto II analyzer (BD Biosciences). The percentage of cells in G0/G1, S and G2/M phases were determined using Modfit 3.0 software.
For cell cycle analysis using 5-bromo-2′-deoxyuridine (BrdU) incorporation, cells were labeled with 10 μM BrdU for 30 min, washed twice with PBS, collected and harvested as above. Cells were pelleted and incubated in 1 mL of 2N HCl containing 0.5% (v/v) Triton X-100 for 30 min then pelleted and washed in 1 mL of 0.1M Na₂B₄O₇.10H₂O (pH 8.5). Cell pellets were sequentially incubated for 30 min with anti-BrdU and Alexa Fluor 488 anti-mouse IgG antibodies (Supplemental methods, Table 2) in PBS containing 2% FBS and 0.5% Tween-20. Cells were washed with PBS- 2% FBS then incubated in RNaseA containing 10 mg/mL PI solution at 37°C for 15 min. Cells were analyzed on the FACSCanto II and cell-cycle analysis was performed using FCS Express software (De Novo, Los Angeles, CA, USA).
For Annexin-V analysis cells were stained with Annexin-V-APC (BD Pharmigen 550474) and 10 μg/mL PI and analyzed with the BD FACSCanto II. Flow cytometry data was analysed with FCSExpress software.

To detect apoptotic cells, both adherent and floating cells were collected and washed twice with PBS. Cells were resuspended in 1× binding buffer (Becton–Dickinson) and stained with 5 µL annexinV-APC (Becton–Dickinson) and 5 µL 7-AAD (Becton–Dickinson) for 15 min in the dark. Samples were analyzed with the FACSCalibur flow cytometer (Becton–Dickinson). Data analysis was performed using CellQuest Pro software (Becton–Dickinson).
To detect viable cells, the activity of the mitochondrial dehydrogenase was determined using the Cell Proliferation Reagent WST-1 in 96-well format (Roche, Mannheim, Germany). To determine caspase-9 activity, Caspase-Glo^® 9 Assay (G8210, Promega) was used. Absorption was measured using the Synergy 2 Multi-Mode Microplate Reader (BioTek).