Figure 1.
Alamar blue fluorescence development following culture with schistosomula.
Wells contained 0, 100 (500/ml) or 1000 (5000/ml) schistosomula. # Wells containing schistosomula but no Alamar blue at time 0 (these values did not alter with time in culture). * P<0.01, ** P<0.0001 relative to wells with no schistosomula added. The mean ± standard deviation of fluorescence, expressed in arbitrary units (AU), is based on 5 replicate wells.
Figure 2.
Comparison of microscopy and Alamar blue to assess activity of known anti-schistosome drugs.
Replicate 96-well microtitre plates were set up containing DMSO at 0.5% and either 10 µg/ml (Figure A) or 1 µg/ml (Figure B) of Oxamniquine (OX), Praziquantel (PZ), Meclonazepam (MCZ), Clonazepam (CZ), Ro15-5458 (Ro15), Oltipraz (OPZ) or Dihydroartemisisin (DHA). 100 schistosomula were added per well. Triplicate wells were set up for each treatment. The % dead or damaged schistosomula were counted on each of days 1, 3, 5 and 7. Alamar blue was added on these days and fluorescence read in Arbitrary Units (AU) 24 h later. Mean fluorescence values from wells containing AB and cM169 without parasites were subtracted from the values for test wells. The results for each of the triplicate wells are shown in the graphs with the horizontal bars representing the mean values. The dotted horizontal line on each AB graph corresponds to an AU value 25% lower than the mean for the triplicate DMSO controls. Values for medium alone controls were very comparable to the DMSO values (data not shown).
Figure 3.
Comparison of microscopy and Alamar blue to assess activity of compounds previously screened as active or inactive against adult schistosomes in vitro.
See Legend to Figure 2 for most details. A: 10 µg/ml, B: 1 µg/ml. “Adult +ves”: 33 compounds previously found to be lethal to adult worms in vitro. “Adult −ves”: 30 compounds found to be inactive against adult worms in vitro.
Figure 4.
Comparison of microscopy and Alamar blue assays during routine screening.
See Legend to Figure 2 for most details. Results are shown for the screening of 558 compounds (i) by microscopy at day 3 of culture, larvae being reported as DEAD, morphologically DAMAGED or NORMAL and (ii) by addition of Alamar blue on day 7 and reading 24 h later.
Figure 5.
“Crystal-like” formation on schistosomula.
An example of crystal-like deposit from one of the wells containing schistosomula categorized as DAMAGED from Figure 4. (Magnification ×100). Image captured using a Leitz DMRB microscope (Leica, Germany) with attached Leica DFC 420 digital camera controlled by Leica Application Suite version 3.1 software (Leica Microsystems [Switzerland] Ltd.).