FACS-based assay to assess parasite growth inhibition 48 hr Measured in Cell-Based System Using Flow Cytometry - 2126-03_Inhibitor_Dose_DryPowder_Activity
The assay used flow cytometry to measure growth inhibition of the malaria parasite, Plasmodium falciparum. Human red blood cells infected with P. falciparum parasites were identified based on staining with SYBR green, which fluoresces when intercalated with nucleic acids. Since mature human erythrocytes lack nuclei and RNA, only parasitized red blood cells stain positive for SYBR green. In addition, viable parasites were distinguished from non-viable parasites using the vital dye MitoTracker Deep Red, which is only retained within metabolically active mitochondria. ..more
BioActive Compounds: 6
Keywords: Plasmodium falciparum, flow cytometry, mitochondria, growth inhibition assay
The assay used flow cytometry to measure growth inhibition of the malaria parasite, Plasmodium falciparum. Human red blood cells infected with P. falciparum parasites were identified based on staining with SYBR green, which fluoresces when intercalated with nucleic acids. Since mature human erythrocytes lack nuclei and RNA, only parasitized red blood cells stain positive for SYBR green. In addition, viable parasites were distinguished from non-viable parasites using the vital dye MitoTracker Deep Red, which is only retained within metabolically active mitochondria.
Parasitized erythrocyte cultures were treated with the test compounds for 48 hours. Control wells lacking drug were used to determine maximal growth of P. falciparum in human erythrocytes for the purposes of deriving IC50 values. SYBR Green I and Mitotracker Red were added at the time of parasite collection prior to flow cytometry.
Expected Outcome: This assay allows for the discovery and characterization of antimalarial compounds. Parasite growth inhibition is measured based on the number of parasites surviving in the compound treated wells relative to the number of parasites surviving in untreated wells. Viable parasites stain positive for SYBR green and MitoTracker Deep Red, while non-viable parasites demonstrate lower MitoTracker staining and uninfected erythrocytes do not retain either dye.
P. falciparum cultures were grown at a 4% hematocrit in type AB donor blood. The red blood cells were diluted in RPMI media (GIBCO) supplemented with 5mg/ml Albumax (GIBCO), 50ug/ml hypoxanthine (Sigma-Aldrich), 2.4 mg/ml Na Bicarbonate (GIBCO), and 10 ng/ml gentamicin (GIBCO). Parasites were incubated at 37 degrees C, in a humidified chamber (Billups-Rothenberg, Inc), gassed with 5%CO2/5%O2/90%N2.
Two P. falciparum strains were used in these experiments, the chloroquine sensitive 3D7 strain, and the chloroquine resistant Dd2 strain. Assays were conducted in flat bottom 96-well plates in a 200 ul culture volume, with red blood cells (AB blood obtained from anonymous donors via Interstate Blood Bank, Inc.) at a 1% hematocrit and starting parasitemias of 0.1%. Compound dilutions were dissolved in DMSO. They were tested at 10 five-fold dilutions, spanning the 50% inhibition range. At 48 hrs, we suspended the cultures and transferred 20ul of the culture to round bottom 96-well plates. The samples were stained with 40 ul of 2.5x SYBR-green-1 (Invitrogen) and 150 nM MitoTracker Deep Red (Invitrogen) diluted in normal saline dextrose (0.9% NaCl, 5% dextrose) or in complete media. The infected erythrocytes were cultured with the stain for 40 minutes at 37 degrees C. Samples were washed and resuspended in 100 ul of saline dextrose containing 5% fetal calf serum. Samples were assayed on an Accuri C6 cytometer equipped with a Hypercyt microplate sampler, which was able to process a 96-well plate in about 15 minutes. Approximately 80,000 events were collected for 48 hour time points and 30,000 events for 96 hour time points.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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 (10)(Max_Concentration).
No normalization was applied to the raw data signals.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
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.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: RBCs
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)