Fc500 mpl cytometer

We used green fluorescent protein (GFP)-encoding dengue virus propagative vector (DENV_GFP) as representative of the flavivirus genus. The plasmid used to obtain the DENV_GFP vector (pFK-DVs-G2A) was provided by Ralf Bartenschlager (Heidelberg University, Heidelberg, Germany) and Laurent Chatel-Chaix (Institut national de la recherche scientifique, Québec, QC, Canada) [63 (link),64 (link)]. For DENV_GFP, viral titer was measured by plaque assay in VeroE6 cells, as described by [65 (link)]. Briefly, Huh7 cells/well (kindly provided by Pr Hugo Soudeyns) were plated in 96-well plates at 37 °C overnight, treated with plant extracts and controls, i.e., DMSO, H₂O and lycorine (a known anti-DENV alkaloid [66 (link),67 (link)] and infected with at a multiplicity of infection (MOI) of 0.1. Cells were incubated at 37 °C and 5% CO₂ for 48 h. Afterwards, the cells were fixed in 3.7% formaldehyde and the percentage of infection was measured by flow cytometry with a FC500 MPL cytometer (Beckman Coulter, Inc., Brea, CA, USA). Data analysis was performed using the Flowjo software (BD, FlowJo LLC, Ashland, OR, USA).

Briefly, 7.5 × 10³ Huh7 cells/well or 2 × 10⁴ THP-1 cells/well were plated in 96-well plates at 37 °C for 24 h. The next day, norbelladine and its derivatives, as well as the DMSO-dissolved lycorine (Huh7) or DMSO-dissolved raltegravir (THP-1), were serially diluted by a factor of 2 in DMEM or RPMI medium, respectively. Each dilution was added to the cell plates to obtain final concentrations of 1.56 µM to 200 µM for alkaloids and matched concentrations of DMSO, and 0.05 µM to 6.4 µM for lycorine or 0.078 µM to 10 µM for raltegravir. Lycorine and raltegravir were used as DENV and HIV-1 inhibitor controls, respectively [18 (link),55 (link)]. DENV_GFP and HIV-1_GFP were then added at multiplicities of infection (MOIs) of 0.025 and 0.1, respectively. Plates were placed in an incubator at 37 °C and 5% CO₂ for 72 h. Afterward, the cells were fixed in 3.7% formaldehyde and the percentage of infection was measured using flow cytometry with an FC500 MPL cytometer (Beckman Coulter, Inc., Brea, CA, USA). Data analysis was performed using Flowjo software (BD, FlowJo LLC, Ashland, OR, USA). All experiments were performed at least twice. EC₅₀ values were calculated using QuestGraph IC50 calculator software (MLA Quest Graph™ IC50 Calculator. AAT Bioquest, Inc. https://www.aatbio.com/tools/ic50-calculator (accessed on 1 July 2022)).

The antiretroviral activity of P. trianthum Herb.’s crude extract was evaluated using pseudotyped HIV-1_GFP in THP-1 cells. THP-1 cells were seeded at 2.0 × 10⁴ cells per well in 96-well plates and incubated overnight. Cells were treated with 5 concentrations of extract (from 0.1 to 1.56 µg/mL) and then infected with HIV_GFP at a MOI at 1. After 72 h, the percentage of infected cells was measured using a FC500 MPL cytometer (Beckman Coulter, Inc., Brea, CA, USA) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). Matched concentrations of DMSO were used as a negative control. All assays were performed in triplicate.

The anti-HIV-1 activity of compounds 1–5 was evaluated using VSV-G pseudotyped NL43_GFP infection of human monocytic THP-1 cells. THP-1 and NL4–3_GFP were generously provided by Lionel Berthoux and Amita Singh and are described in Ka et al. (2021) [13 (link)]. THP-1 cells were seeded at 2.0 × 10⁴ cells per well in 96 well-plates. The next day, cells were treated with 4 concentrations of each compound (12.5, 25, 50 and 100 mM) and then infected with HIV_GFP at a MOI of 1. After 72 h, cells were stained with propidium iodide (PI, 0.5 mg/mL) and both PI⁺ and HIV-1_GFP⁺ infected cells frequencies were assessed on a FC500 MPL cytometer (Beckman Coulter, Inc., Mississauga, ON, Canada) and analyzed using FlowJo software (FlowJo LLC, BD Biosciences, Ashland, OR, USA). Matched concentrations of dimethyl sulfoxide (DMSO) were used as negative controls. All infection assays were performed in triplicate.

BT-474 cells (1–100 × 10⁶ cells/ml) were cultured in CM with the addition of (i) the DMSO solvent only (Control), (ii) 30 μg/ml of cardanol and (iii) 0.5 μg/ml of doxorubicin (positive control) for 24, 48 and 72 h and then harvested and washed as above. The cell pellet was resuspended and fixed in 500 μl of cold PBS and 200 μl of 70 % (v/v) ethanol at −20 °C overnight or on ice for 4 h. Cells were then harvested and washed as above and the pellet resuspended in 250 μl of PBS with 0.1 mg/ml RNAse and incubated at 37 °C for 30 min. After harvesting, the cells were resuspended in 12.5 μl of 1 mg/ml PI and incubated at RT in the dark for 30 min before being analyzed by flow cytometry on a FC 500 MPL cytometer (Beckman Coulter) recording 2 events per sample. The obtained linear fluorescence profile was interpreted in terms of the (1) sub G₁ phase (apoptotic cells), (ii) G₁ phase (diploid chromosome content), (iii) S phase (DNA synthesis) and (iv) G₂/M subphase (double diploid).