Annexin 5 conjugated to fitc

Supernatant was collected from the sample wells to preserve detached cells. The wells were washed twice with PBS and TrypLE Express Enzyme (Gibco) was added to the wells. The plate was incubated at 37°C for 5 min to allow for detachment of adherent THP-1 macrophages. The floating and harvested cells were combined and washed twice with PBS. Cells were stained with Annexin V conjugated to FITC (eBioscience, San Diego, CA) according to manufacturer’s protocol and flow cytometric analysis was used to measure apoptotic cells.

Cell culture supernatants were collected to preserve detached cells. Cells were washed with twice with PBS and TrypLE Express enzyme was added to the wells. The plate was incubated at 37°C for 5 min to allow for through detachment of adherent cells. Harvested cells were washed twice with PBS and stained with Annexin V conjugated to FITC (e-Biosciences, San Diego, CA) in combination with Fixable Viability Stain 780 according to the manufacturer’s protocol. Flow cytometric analysis was then used to quantify the amount of healthy (Annexin V-, FVS780-), apoptotic (Annexin V+, FVS780-), and necrotic cells (Annexin V-, FVS780+ and Annexin V+, FVS780+).