Antiplasmodial activity was assessed by SYBR green I fluorescence, as described previously.33 Twofold serial dilutions of compounds were incubated with Plasmodium falciparum cultures at 1% parasitemia and 1% hematocrit in 96-well plates (Santa Cruz Biotechnology, Dallas, TX) at 37 °C under a 5% CO2 humidified atmosphere. The maximum DMSO concentration in assays was 0.1%. DMSO and CQ were used as growth and inhibition controls, respectively. After 72 h of incubation, plates were frozen at −80 °C, thawed, and incubated with 100 μL of lysis buffer (20 mM Tris-HCl, 0.008% saponin, 5 mM EDTA, 0.08% Triton X-100, and 0.01% SYBR Green I). Following incubation in the dark at room temperature for 1 h, the fluorescence signal was measured at 485 nM excitation and 530 nM emission using a Synergy Neo2 multimode reader (BioTek, Winooski, VT, USA). GraphPad Prism 8.0 software was used to generate nonlinear dose–response curves and calculate the EC50 values for each compound.
96 well plates
Manufactured by Santa Cruz Biotechnology
Sourced in United States
96-well plates are a common laboratory equipment used for various experimental procedures. They consist of a flat, rectangular plate with 96 individual wells, arranged in a 8 rows by 12 columns grid. The wells are designed to hold small volumes of liquid samples or reagents. These plates are typically made of plastic and are available in different materials, such as polystyrene or polypropylene, to suit different experimental requirements.
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3 protocols using 96 well plates
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Antiplasmodial Activity Evaluation by SYBR Green I
2
Anti-Plasmodium Compound Screening
3
Cytotoxic Effects of P. lanceolata on Breast Cancer Cell Lines
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crystal violet cell viability assay was employed to measure the cytotoxic effect of the plant extract. Human breast cancer cell lines (MDAMB, AMJ13, MCF7, and CAL51 cells) and normal mouse embryonic cells (MEF) were seeded at a density of 7000 cells/well in 96-well plates (Santa Cruz Biotechnology, USA). After 24 h of incubation or after a confluent monolayer was formed, the cells were treated with P. lanceolata leaf extract at 2-fold dilutions (4000, 2000, 1000, 500, 250, 125, 62.5, 31.25 μg/ml to 15 μg/ml) in culture media. The assay was performed in triplicate. Cell viability was determined after 72 h of treatment by cell staining with 50 μl of crystal violet (Sigma Aldrich, USA) followed by incubation at 37 °C for 2 h. The stain was aspirated, and PBS was used to wash the wells. A microplate reader (Biochrom, UK) was used to measure the absorbance at 492 nm.
Results of the assay were shown as percentage of proliferation relative to control cells [21] .
Results of the assay were shown as percentage of proliferation relative to control cells [21] .
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