Cellometer vision cba image cytometry system

Cell based apoptosis assay was performed using Cellometer Vision CBA Image Cytometry System (Nexcelom Bioscience LLC, Lawrence, MA) with two fluorophore Annexin V-FITC and Propidium Iodide (PI) staining solution, according to manufacturer’s instructions. Briefly, cells were harvested using trypsin, then spin down at 300g for three minutes and pellets were washed with 1XPBS, cells were counted using hematocytometer. Collected 100,000 to 150,000 cells and cells/pellet was resuspended in 40μl of Annexin V binding buffer. 5μl each of Annexin V –FITC reagent (green) and PI (red) were added to binding buffer containing cells; gently mix solution by pipetting up and down ten times, then incubate for 15 min at RT in the dark; after incubation, add 250μl of 1XPBS and spin down at 300g for three minutes, then re-suspended the cell pellets in 50μl of Annexin V binding buffer, then assess the cells apoptosis. Gate purple represents live cells, gate green represents the positive apoptotic cells, gate blue represents for the detection of positive necrotic cells and gate red represents debris.

Cell survival was determined with Annexin V-FITC/PI apoptosis assay using Cellometer Vision CBA Image Cytometry System (Nexcelom Bioscience, Lawrence, MA), as described previously [30 (link), 31 (link)]. In brief, HT22 cells from different transfected groups were trypsinized, collected, resuspended in 1x PBS, and counted. Cells (1.0–1.5 × 10⁵ cells) were then resuspended in 40μl of Annexin V binding buffer and processed according to the manufacturer’s instructions. Finally, cell pellet was resuspended in Annexin V binding buffer and counted using Cellometer [30 (link), 31 (link)].

A cell-based apoptosis assay was performed, using the Cellometer Vision CBA Image Cytometry System (Nexcelom Bioscience, LLC, Lawrence, MA, USA) with 2 fluorophores-Annexin V-FITC and propidium iodide (PI) staining solutions, following manufacturer’s instructions. Briefly, neuroblastoma cells were harvested using trypsin, then spun down to 300 g for 3 min. The pellets were washed with 1×PBS. Cells were counted using a hematocytometer, and 100,000 to 150,000 cells were collected and resuspended in 40 μl of an Annexin V binding buffer. Five μl of Annexin V-FITC reagent (green) and PI (red) each were added to the binding buffer containing the cells. The solution was gently mixed by pipetting up and down. It was then incubated for 15 min at room temperature in the dark. After incubation, 250 μl of 1×PBS was added and spun down at 300 g for 3 min. Cell pellets were re-suspend in 50 μl of an Annexin V binding buffer and assessed for apoptosis analysis [32 (link)].