Cell-based secondary assay to test the inhibitory activity of small molecule on Plasmodium flaciparum (HB3 strain) survival in red blood cells Measured in Cell-Based System Using Plate Reader - 2120-06_Inhibitor_Dose_CherryPick_Activity
Assay Overview: This assay has been performed in collaboration with Dr. Amanda Lukens from Dyane Wirth's laboratory at the Harvard School of Public Health, Boston MA. Plasmodium falciparum has the capacity to infect red bllod cells and use the transcriptional and translational machinery in the host cell to replicate. In the assay, we are testing the ability of a small molecule to kill the more ..
BioActive Compounds: 247
Keywords: Plasmodium falciparum, malaria, Hsp40
Assay Overview: This assay has been performed in collaboration with Dr. Amanda Lukens from Dyane Wirth's laboratory at the Harvard School of Public Health, Boston MA. Plasmodium falciparum has the capacity to infect red bllod cells and use the transcriptional and translational machinery in the host cell to replicate. In the assay, we are testing the ability of a small molecule to kill the parasite Plasmodium falciparum HB3 strain present inside red blood cells. The viability of the parasite was determined by measuring the fluorescence emitted by the interacton the green-based fluorescent dye SYBR with the parasite DNA content detect present inside the host cells. Mefloquine, a known antibiotic able to Plasmodium falciparum, was used as positive control.
Expected Outcome: Compounds decreasing the fluorescent signal normalized to the positive control more than 35% (AbsACNN-35) at a concentration below 10uM will be considered actives.
SYBR Drug Assay Standard Protocol Plasmodium falciparum HB3 strain
- cultures are maintained and the assay performed in RPMI media supplemented with Albumax II (recipe below)
- stock cultures are maintained at 5% hematocrit
- cultures are maintained in incubators at 37 degrees C and 4.1% CO2 + 1%O2 + balance N2 gas environment
Day -2Synchronize cultures for assay
1.Make Giemsa slide of seed culture(s) and determine total parasitemia and ring parasitemia
- cultures that are minority rings are difficult to effectively synchronize
- cultures >3 days post-thaw can be used; don't use cultures > 8 weeks from thaw date
2.Calculate volume of culture to synchronize according to percentage of rings and hematocrit
3.Set up a 0.75-1% ring culture of appropriate volume
- don't use blood older than three weeks or culture medium thawed for more than a week
- pellet RBCs by centrifugation
- remove supernatant and resuspend in 40% original culture volume of 5% pre-warmed sorbitol solution (i.e. for a 25mL culture, add 10mL sorbitol)
- incubate cells in sorbitol at 37 degrees C or room temperature for 10-20 minutes
- centrifuge cells and remove sorbitol containing supernatant
- resuspend cells in an appropriate volume of complete media for a 5% hematocrit culture and return to incubator
Day -1 Feed cultures
1.Make a slide of each synchronized culture to check synchronization and parasitemia
- if trophozoite parasitemia is >2%, split culture to 1% trophs and replace media; otherwise, just replace media on culture
2.Make sure sufficient blood and complete media are available for assay tomorrow
3.Calculate drug dilutions, label plates and organize equipment for assay tomorrow
Day 0 Set up assay
1.Feed cultures on morning of assay; prepare and warm media for assay
2.Prepare Plate Map and HTS Excel files for assay
3.Make Giemsa slides and determine total and ring parasitemias
- parasitemias should be between 2-7% with 90% of parasites at ring stage
4.Harvest 5mL sample for RNA
- centrifuge to obtain red cell pellet
- lyse RBCs with 0.15% saponin solution
- transfer lysate to a microfuge tube and centrifuge at max speed for 5 minutes
- remove supernatant and resuspend pellet in 250uL of buffer RTL from Qiagen RNAeasy Mini Kit
- store sample at -80 degrees C for later RNA extraction
5.Determine hematocrit of culture
6.Thaw compounds and prepare 96-well drug dilution and control plates
7.Transfer drug dilutions with Velocity robot to 384 well plates
8.Make cell suspension for dispensing into assay plates
- prepare a 1% parasitemia/1.3% hematocrit solution for the assay plates (final = 1% parasitemia/1%hematocrit in assay plate)
9.Dispense 30 ml cell suspension into 384 plate using Thermo Multidrop with sterilized cartridge
- gently swirl bottle of cell suspension often to ensure homogenous mixture dispensed across all plates
- gently tap plates after additon of solution
10. Transfer plates to incubator maintained at 37 degrees C, 4.1% CO2 + 1%O2 + balance N2, and incubate for 72 hours.
- can stack plates two-four high in incubator
Day 3 Stain plates with SYBR
1.After 72 hour incubation, make Giemsa slide from control plate and determine parasitemia. (2 slides)
- parasitema should be between 5-12 %, with the majority of parasites in trophozoite or early shizont stage
2.Warm appropriate aliquot of SYBR buffer and SYBR Green I to room temperature
3.Add SYBR to buffer (1:1000 dilution) and mix.
- wrap bottle in foil to prevent exposure of SYBR containing solution to light
4.Dispense 10 ml SYBR/buffer into 384 plate using Thermo Multidrop with cartridge reserved for SYBR
5.Seal plates with foil seals; tear off seal tab and press seal firmly.
6.Spin plates at 1000 rpm for 1 minute
7.Incubate plates overnight at room temperature
8.Rinse Multidrop cartridges and sterilize
- Rinse cartridge for 1 min with 1% detergent solution, followed by distilled water, then fill cartridge with 70% ethanol, let stand for 5 min, then purge
9.Filter SYBR-contaminated waste through charcoal filter and discard down drain
Day 4 Read plates
1.Spin plates at 2000 rpm for 10 minutes.
2.Read plates in Wallac plate reader.
Complete RPMI media* (per 1L):
10.44g RPMI 1640
2.016g sodium bicarbonate
5g Albumax II
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -35.0
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)