Glucosamine glcn

For asynchronous growth assays of PfHsp70x-DDD lines, parasites were washed twice and incubated without TMP. For asynchronous growth assays of PfHsp70x-glmS and PfHsp70x-M9 parasites, 5 or 10 mM glucosamine (GlcN) (Sigma) was added to the growth medium. Asynchronous growth assays of PfHsp70x-KO parasites were performed in medium containing WR99210. Parasitemia was monitored every 24 h via flow cytometry. For flow cytometry, aliquots of parasite cultures (5 μl) were stained with 1.5 mg/ml acridine orange (Molecular Probes) in phosphate-buffered saline (PBS). The fluorescence profiles of infected erythrocytes were measured by flow cytometry on a CyAn ADP (Beckman Coulter) or CytoFLEX (Beckman Coulter) instrument and analyzed by FlowJo software (Treestar, Inc.). Whenever required, parasites were subcultured to avoid high parasite density, and relative parasitemia at each time point was back-calculated based on actual parasitemia multiplied by the relevant dilution factors. One hundred percent parasitemia was determined as the highest relative parasitemia and was used to normalize parasite growth. Data were fit to exponential growth equations using Prism (GraphPad Software, Inc.).