Verapamil hydrochloride (VP; Sigma-Aldrich, Australia), mibefradil hydrochloride (MF; Tocris Bioscience, UK), L703,606 (L7; Sigma-Aldrich, Australia), and primaquine (PQ; World Wide Antimalarial Resistance Network [WWARN] QA/QC Reference Material Programme [42 (link)]) were selected as CQ resistance reversal agents (CQRRAs) in this study. The intrinsic antimalarial activities of the recently reported MF and L7 had hitherto been tested only in the CQr strain K1 (28 (link)). Therefore, prior to the experiments in field isolates, the intrinsic antimalarial properties of these reversal agents, in addition to those of VP and PQ, were assessed in CQr (K1 and W2) and CQs (3D7 and FC27) P. falciparum laboratory strains (BEI Resources, ATCC, Manassas, VA, USA).
Verapamil hydrochloride vp
Manufactured by Merck Group
Sourced in Australia, United States
Verapamil hydrochloride (VP) is a white, crystalline powder. It is commonly used as a laboratory standard and reference material for analytical and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
P falciparum, by BEI Resources (1 mentions)
Primaquine, by Merck Group (1 mentions)
Rhod 123, by Merck Group (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
7 aminoactinomycin d 7 aad, by Merck Group (1 mentions)
Forma series 2 water jacket, by Thermo Fisher Scientific (1 mentions)
Fumitremorgin c, by Merck Group (1 mentions)
3 protocols using verapamil hydrochloride vp
1
Evaluating Antimalarial Reversal Agents
2
Rhod-123 Efflux Assay in Leishmania
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The efflux of the fluorescent probe Rhod-123 (Sigma-Aldrich Inc., St. Louis, MO, USA) was tested in the LaWT and LaSimR strains using a cytometer (CytoFLEX S Beckman Coulter). First, promastigotes of L. amazonensis (5 × 106 parasites/mL) in the log-phase of growth were incubated in RPMI medium in the presence and the absence of 100 μM of verapamil hydrochloride (Vp) (Sigma-Aldrich Inc., St. Louis, MO, USA), an inhibitor of the drug efflux pump P-glycoprotein, for 1 h at 26 °C. Next, parasites were incubated in the presence or absence of Rhodamine-123 (Rhod-123) (5 μg/mL) for 30 min [35 (link),36 (link)]. The parasites were then washed three times, resuspended in 1 mL PBS buffer, and incubated in the presence or absence of Vp (100 μM) for 30 min and 90 min, in triplicates, to measure Rhod-123 efflux. Data analysis was performed using the CytExpert software.
3
Multiparametric Flow Cytometry of Side Population
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Vybrant DyeCycle Violet Ready Flow™ Reagent (Invitrogen) was added to cells in phenolfree DMEM:F12 medium and incubated at 37 o C in a 5% CO2 cell culture chamber (Forma Series II Water Jacket; ThermoFisher Scientific) for 30 minutes. The concentration of DCV was tested at both 1X and 2X, and based on these experiments (Fig S1), all future experiments used the concentration of 2X, or 160uL in 10 6 cells/ml.
For experiments involving ABC transporter inhibition, fumitremorgin C (FTC; Sigma) and (±) verapamil hydrochloride (VP; Sigma) were added to unwashed cells at final concentrations of 10uM and 50uM, respectively, after DCV incubation and kept in the same conditions for additional 30 minutes. Cell were then kept on ice in dark until sort, and 7-Amino-Actinomycin D (7AAD, 40 ug/ml, Sigma) was added to cell suspensions 10 minutes before analysis for dead cell discrimination. For experiments determining the identity of the side population, CD31 antibody conjugated to allophycocyanin (APC), BD Biosciences, BioLegend), was added to cells in DMEM:F12 at final concentration of 1:50 (24) and incubated on ice in the dark for 30 minutes before DCV incubation, which followed the same workflow as discussed above. Antibodies, dyes, and drugs used for all experiments are listed in Table 1.
For experiments involving ABC transporter inhibition, fumitremorgin C (FTC; Sigma) and (±) verapamil hydrochloride (VP; Sigma) were added to unwashed cells at final concentrations of 10uM and 50uM, respectively, after DCV incubation and kept in the same conditions for additional 30 minutes. Cell were then kept on ice in dark until sort, and 7-Amino-Actinomycin D (7AAD, 40 ug/ml, Sigma) was added to cell suspensions 10 minutes before analysis for dead cell discrimination. For experiments determining the identity of the side population, CD31 antibody conjugated to allophycocyanin (APC), BD Biosciences, BioLegend), was added to cells in DMEM:F12 at final concentration of 1:50 (24) and incubated on ice in the dark for 30 minutes before DCV incubation, which followed the same workflow as discussed above. Antibodies, dyes, and drugs used for all experiments are listed in Table 1.
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