Multiflo dispenser

To quantify the rate
of false positives reported during the PbLuc liver stage reconfirmation
screen, hits selected for tertiary round screening (405 compounds
from fresh DMSO stocks) were also tested for luminescent interference
between recombinant firefly luciferase and luciferin. Initially, 40
nL of selected compounds was dispensed using a Gen 4 Plus Acoustic
Transfer System (Biosero, San Diego, USA) at 1:3 in 12-point dose
response format (25 to 141.13 × 10^–6 μM;
final DMSO concentration of 0.5% per well) in 1536-well, white, opaque-bottom
plates (ref# 789173-F, Greiner Bio-One). A 24-point single concentration
series of Luciferase Inhibitor-II (9.8 μM per well) was dispensed
as a positive control, while 96 wells of DMSO at 0.5% per well acted
as the negative control. Separately, a solution of 20 pM recombinant
luciferase (Promega cat# E170A) was prepared on ice by successive
dilutions (first 1:999 then into final solution at 20 pM) in phenol
naive DMEM (Invitrogen cat# 31053-028), 5% FBS (Corning), and 5×
Pen Strep Glutamine (Invitrogen). This solution was dispensed at 8
μL per well into the previously spotted plates using a MultiFlo
dispenser (BioTek) and incubated at room temperature for 1 h prior
to the addition of 1 μL of BrightGlo (Promega). Immediately
after the addition of BrightGlo, plates were read using the EnVision
Microplate reader (PerkinElmer).

Prior to parasite addition,
10 nL (final concentration of 2 μM) of each compound was dispensed
using an Echo 550 acoustic dispenser (Labcyte) in 1536-well, black,
clear-bottom plates (ref# 789866-691-2B, Greiner Bio-One). High-content
imaging experiments were performed on stage V gametocytes as previously
described.^{51 (link)} Briefly, mature gametocytes
were diluted to 0.5–0.75% gametocytemia and 1.25% hematocrit
in serum free screening media. Cultures were dispensed at 10 μL
per well using a MultiFlo dispenser (BioTek) and incubated at 37 °C
for 72 h (3% O₂, 5% CO₂, and 92% N₂). A solution of 2.5 μM MitoTracker Red CMXRos and 0.13% saponin
(w/v) was made in screening media and added to each well for 1–2
h at 37 °C to allow for complete red blood cell lysis. The Operetta
(PerkinElmer) high content imaging platform was used to read all 1536-well
plates with the analysis performed by the accompanying Harmony software. Compounds with >50%
inhibition were resourced
from younger stocks and plated in 10-point dose response format. All
screening and analysis steps were then repeated as previously described.