Novocyte penteon flow cytometer system

In this work, we demonstrated a cytometric analysis that allowed us to multiparametrically assess three markers of cell health: changes in the mitochondrial potential, expression of phosphatidylserine on the cell surface, and membrane permeabilization. The use of reagents MitoSense Red, annexin V, and 7-AAD in the Millipore FlowCellect™ MitoDamage Kit allowed us to gain information on early, mid, and late apoptosis with one simple assay. Cells were treated with the synthesized compounds at a concentration of 0.5 µM and incubated at 37 °C for 4 h. After this time, the cells were dissociated using an accutase solution, stained, and analyzed using the NovoCyte Penteon Flow Cytometer System (Agilent, 5301 Stevens Creek Blvd., Santa Clara, CA, USA), according to the manufacturer’s protocols for the FlowCellect™ MitoDamage Kit and FlowCellect™ Oxidative Stress Characterization Kit (Merck).

Viability (live/dead) assessment was performed by staining cells with 7-AAD (7-aminoactinomycin D; Biolegend). After treatment, cells were harvested, washed 1–2 times with phosphate-buffered saline (PBS), and centrifuged at 400× g for 5 min. Cell pellets were resuspended in 200 µL of flow cytometry staining buffer (PBS without Ca²⁺ and Mg²⁺, 2.5% FBS) and stained with 1 mM/L of 7-AAD staining solution for 15 min at room temperature in the dark. Samples were assessed using the NovoCyte Penteon Flow Cytometer System (Agilent, 5301 Stevens Creek Blvd., Santa Clara, CA, USA). Detection of 7-AAD emission was collected through a 675/30 nm filter in the FL4 channel.

Quantification of cytochrome c release from mitochondria in apoptotic cells was presented to detect the mitochondrial pathway of apoptosis in cells using flow cytometry with the FlowCellect™ Cytochrome c Kit (FCCH100110). For this, cells were treated with FITC-conjugated antibodies against cytochrome c and control anti-IgG1-FITC, together with the use of an optimized fixation procedure, permeabilization, and blocking buffer (Luminex^®, USA). Higher levels of cytochrome c fluorescence were observed in living cells, while lower levels were characteristic of apoptotic cells in which cytochrome c was released from mitochondria into the cytoplasm. Jurkat cells were incubated for 4 h with the synthesized substances at a concentration corresponding to their 24-h CC50 in a culture plate and assessed using the NovoCyte Penteon Flow Cytometer System (Agilent, 5301 Stevens Creek Blvd., Santa Clara, CA, USA). The obtained data were processed using NovoExpress^® software (ACEA).