Lsr fortessa facs analyzer

Extracts from Passiflora edulis fruit (PEF), Annona muricata leaves (AML), Annona muricata seeds (AMS) that displayed the best cytotoxicity as well as doxorubicin were used to treat CCRF–CEM cells (1 × 10⁶) at their IC₅₀ values. Thus, CCRF–CEM cells were cultured in RPMI medium as described above, in the presence of each sample at a concentration corresponding to the IC₅₀ values obtained in the cell line. The cell cycle was then analyzed after incubation for 24, 48 and 72 h. All reagents, experimental conditions and apparatus were identical to those previously reported (Kuete et al. 2013a (link); Dzoyem et al. 2013 (link)). Briefly, cell cycle analysis was performed by flow cytometry using Vybrant^® DyeCycle™ (Invitrogen, Darmstadt, Germany). Cells were measured after Vybrant^® DyeCycle™ Violet staining (30 min at 37 °C) on a LSR-Fortessa FACS analyzer (Becton–Dickinson, Heidelberg, Germany) using the violet laser. Vybrant^® DyeCycle™ Violet stain was measured with 440 nm excitation. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate.

CCRF-CEM cells were treated with AAR, ACL or vinblastine. The MMP was analyzed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) (JC-1; Biomol, Hamburg, Germany) staining as previously described [19 (link)]. Cells were measured in a LSR-Fortessa FACS analyzer (Becton-Dickinson). The JC-1 signal was measured at an excitation of 561 nm (150 mW) and detected using a 586/15 nm band-pass filter. The signal was analyzed at 640 nm excitation (40 mW) and detected using a 730/45 nm bandpass filter. Cytographs were analyzed using FlowJo software (Celeza, Olten, Switzerland). All experiments were performed at least in triplicate.

To quantify the modification of the mitochondrial membrane potential (MMP) and the reactive oxygen species (ROS) production in CCRF-CEM cells resulting from the application of BTL, either compound 8 or DMSO (negative control), or the respective positive controls for MMP or ROS evaluations, valinomycin or H₂O₂ (Sigma-Aldrich, Taufkirchen, Germany), the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Biomol, Hamburg, Germany) staining and the 2´,7´-dichlorodihydrofluorescein diacetate (H₂DCFH-DA) (Sigma-Aldrich) staining, respectively for MMP and ROS measurements, were combined with the flow cytometry used in similar experimental conditions, as earlier reported [45 (link)]. The concentrations of samples used to treat the CCRF-CEM cells were 0.25, 0.5, 1, and 2 folds the IC₅₀. The treated cells (1 mL; 1 × 10⁶ cells) were incubated for 24 h under the standard cell culture condition. Cells were further stained for 30 min with 10 µL of staining solution (JC-1 for ROS evaluation or H₂DCFH-DA solution for ROS evaluation) according to the manufacturer’s protocol. Using the LSRFortessa FACS analyzer (Becton–Dickinson, Heidelberg, Germany), the amount of 1 × 10⁴ cells was further measured as described earlier [31 (link), 45 (link)–47 (link)].

The effects of extracts on the MMP were analyzed by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) (JC-1; Biomol, Germany) staining (Kuete et al. 2013c (link)). JC-1 is a dye that can selectively enter into mitochondria and exhibits an intense red fluorescence in healthy mitochondria with normal membrane potentials. In cells with reduced MMP, the red fluorescence disappears. Briefly, 1 × 10⁶ CCRF–CEM cells treated at different concentrations with PEF, AML, AMS or vinblastine for 24 h were incubated with JC-1 staining solution according to the manufacturer`s protocol for 30 min. Subsequently, cells were measured in a LSR-Fortessa FACS analyzer (Becton–Dickinson). For each sample, 1 × 10⁴ cells were counted. The JC-1 signal was measured with 561 nm excitation (150 mW) and detected using a 586/15 nm bandpass filter. The samples signal was analyzed with 640 nm excitation (40 mW) and detected using a 730/45 nm bandpass filter. All parameters were plotted on a logarithmic scale. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed in triplicate.

The JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) was applied for the detection of MMP by flow cytometry according to the manufacturer’s instructions. The cationic dye, 5, 5’, 6, 6’- tetrachloro- 1, 1’, 3, 3’- tetraethylbenzimidazolylcarbocyanine iodide (JC-1) enters the mitochondria and changes its fluorescent properties based on the aggregation of the probe. In healthy cells having high MMP, JC-1 forms complexes known as J-aggregates with intense red fluorescence. On the other hand, in cells with low MMP, JC-1 remains in its monomeric form showing green fluorescence [70 (link)]. Aliquots of 5×10⁵ cells/ml were treated with DMSO as negative control, doxorubicin as positive control or 1-, 2- or 4-fold IC₅₀ of Aloe-emodin for 24 and 48 h. A LSR-Fortessa FACS analyzer (Becton–Dickinson) was used to detect the J-aggregate form of JC-1 with an excitation wavelength of 535 ± 20 nm and an emission wavelength of 590 ± 20 nm as well as the monomeric form of JC-1 at excitation and emission wavelengths of 485 and 535 nm, respectively. The results were analyzed by the FlowJo software (Celeza, Olten, Switzerland). 2×10⁴ cells were counted for each experiment which were repeated in triplicate [71 (link)].

Extracts from Albizia adianthifolia roots (AAR) and Alchornea cordifolia leaves (ACL) that displayed the best cytotoxicity as well as doxorubicin were used to treat CCRF-CEM cells (1 × 10⁶) at their IC₅₀ values. The cell cycle was then analyzed after incubation for 24 h, 48 h and 72 h. All reagents, experimental conditions and apparatus were identical to those previously reported [12 (link), 16 (link)]. Briefly, cell cycle analysis was performed by flow cytometry using Vybrant® DyeCycle™ (Invitrogen, Darmstadt, Germany). Cells were measured after Vybrant® DyeCycle™ Violet staining (30 min at 37 °C) on a LSR-Fortessa FACS analyzer (Becton-Dickinson, Heidelberg, Germany) using the violet laser. Vybrant® DyeCycle™ Violet stain was measured with 440 nm excitation. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate.

The modification of the MMP as well as the ROS levels after the application of BAL, and flavonolignan 2 at ¼ IC₅₀, ½ IC₅₀, IC₅₀, and 2 × IC₅₀ to CCRF-CEM cells (1 × 10⁶) was performed by flow cytometry [45 (link)]. DMSO was used as solvent control while valinomycin served as a positive control. The cells were incubated for 24 h and further stained for 30 min with JC-1 [45 (link)] and measured (1 × 10⁴ cells) in an LSR-Fortessa FACS analyzer (Becton–Dickinson) [45 (link)]. For ROS determination, CCEF-CEM cells were similarly treated BAL, the flavonolignan 2, hydrogen peroxide (H₂O₂; positive control), DMSO (solvent control) as in the MMP analysis, then incubated for 24 h. They were further stained with H₂DCFH-DA and measured (1 × 10⁴ cells) in an LSR-Fortessa FACS analyzer [40 (link), 51 (link), 52 (link)].