Glomax multi detection system with instinct software

The Luciferase reporter assay was established to enable accurate, reliable and quantifiable investigations of the stage-specific action of gametocytocidal compounds for each of the early and late gametocyte marker cell lines; NF54-PfS16-GFP-Luc and NF54-Mal8p1.16-GFP-Luc (kind gift from David Fidock, Columbia University, USA) [37 (link)]. Gametocytogenesis was induced on synchronized asexual parasites as described above, with the exception that NAG treatment was initiated from day 1 – 4 to remove asexual parasites early and allow more synchronized early stage gametocytes. Drug assays were set up on day 5 and 10 (representing early stage I/II/III and mature stage IV/V gametocytes, respectively). In each instance, assays were set up in triplicate using a 2 % gametocytaemia, 2 % haematocrit culture and 48 h drug pressure in a gas chamber (90 % N₂, 5 % O₂, and 5 % CO₂) at 37 °C. Luciferase activity was determined in 20 μl parasite lysates by adding 50 μl luciferin substrate (Promega Luciferase Assay System) at room temperature and detection of resultant bioluminescence at an integration constant of 10 s with the GloMax^®-Multi + Detection System with Instinct^® Software.

HEK293 cells were plated in 96 well plates (3.0–4.0 × 10⁴) in antibiotic-free complete growth media (EMEM + 10% FBS) 24 h prior to transfection. Transfections (three separate experiments) were performed in triplicate by adding 10 ul of transfection complex containing 0.5 ul Lipofectamine 2000 (Invitrogen), 100 ng of Bdnf miRNA Target Sequence 3′UTR expression clone (GeneCopoeia), 60 nmol/L miR-15b mimic (Dharmacon), or 37 nmol/L antisense LNA oligo (Exiqon). Experimental conditions included plasmid alone, plasmid + miR-15b mimic, and plasmid + miR-15b mimic and LNA inhibitor. Wells with cells alone were used to subtract background expression when read on the plate reader. Then, 24 h after transfection cells, were lysed and both Firefly luciferase and Renilla luciferase expression was measured using the Luc-Pair miR Luciferase Assay (GeneCopoeia) in a GloMax^®-Multi+ Detection System with Instinct™ Software (Promega). After background subtraction, Firefly luminescence was normalized to Renilla luminescence producing relative luciferase unit (RLU) values. Data are reported as a percent change from the BDNF plasmid alone levels, displayed with 95% confidence intervals. A paired t-test was used to compare the miR-15b mimic groups and miR-15b mimic plus inhibitor groups to BDNF plasmid alone groups. All calculations were done in R.

U87 MG cells were 24 h or 48 h incubated with 1, 10, 20, 50, and 100 µL/mL distilled water, sample 1, sample 2, sample 3, and sample 4 in complete cell culture media. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich, Darmstadt, Germany) assay detected at 560 nm and 750 nm by 96-well plate absorption reader (GloMax^®-Multi+ Detection System with Instinct Software, Promega Corporation, Madison, WI, USA). A standard protocol for MTT assay was performed, i.e., 10 µL of 5 mg/mL MTT dissolved in phosphate saline buffer (PBS, pH = 7.4) were added into each well filled with 100 µL medium (alternatively, 100 µL of MTT were added into 1 mL media in Petri dish), and the plate was 1 h incubated in the dark at 37 °C; after 1 h, the cell culture medium was taken out from the well, and 200 µL (alternatively 1 mL in Petri dish) of dimethylsulfoxide (Sigma-Aldrich, Darmstadt, Germany) was added to each well to make dissolved formazan crystals.