Apc annexin 5 dead cell apoptosis kit with apc annexin 5

Cell viability was detected by trypan blue staining [20 ] that identifies cells in which the cell membrane is compromised. Cells were diluted 1:100 in 0.25 % trypan blue solution (Invitrogen) and counted in a hemocytometer to assess the number of dead blue cells from the total number of cells counted. An additional measure of cell viability was obtained using FACS for Annexin V. After harvesting the AMSCs, cells were centrifuged for 5 min at 1200 rpm.
Apoptotic cells were detected using APC Annexin V/Dead Cell Apoptosis Kit with APC Annexin V and SYTOX® Green (Invitrogen, Eugene, OR, USA). Cells were resuspended at a concentration of 1 × 10⁶ cells/mL in 1X Annexin-binding buffer; 5 μL APC-Annexin V and 1 μL of the 1 μM SYTOX® Green stain working solution were added to each 100 μL cell suspension. The cells were incubated at 37 °C in an atmosphere of 5 % CO2 for 15 min, and then analyzed by flow cytometry [21 (link)]. DNA damage that is associated with cellular senescence and migration were each measured by western blot using, respectively, H2AX antibodies that detect nuclear sites with telomere loss or other DNA damage, and the EMD Millipore (Billerica, MA, USA) QCM™ 24-well chemotaxis cell migration assay (Cat# ECM508) as described [22 (link), 23 (link)].

Apoptosis of swine Obese- and Lean-MSCs was detected using the APC Annexin-V/Dead Cell Apoptosis Kit with APC Annexin-V and SYTOX® Green (Invitrogen, Eugene, OR; Cat.#V35113), as described [42 (link)]. Analysis was performed using a FlowSight (Amnis, Seattle, WA) flow-cytometer and IDEAS (Amnis, v6.0).
To assay cellular response to challenge, swine Obese- and Lean-MSCs were treated for 24 h in vitro with pro-apoptotic staurosporine dissolved in DMSO (Cat.#S6942, Sigma-Aldrich, final concentration 20 nM) [56 (link)], or with DMSO alone (0.1% v/v); the final solution was filtered through a 0.22 µm PVDF syringe filter before use. Apoptosis was then evaluated by terminal deoxynucleotidyl transferase dUTP nick-end-labeling (TUNEL) assay using the DeadEnd™ Fluorometric TUNEL System (Promega), with TUNEL-positive cells counted in a blinded fashion in an average of 7 fields/sample under fluorescence microscopy at 40X magnification and normalized to the number of DAPI-stained nuclei.