Coulter epics elite flow cytometer

After ZD6474 treatment for 24 hours, cells were harvested, washed three times with ice-cold PBS, then fixed by resuspension in 75% ice-cold methanol/PBS and incubated overnight at 4°C. After fixing, samples were pelleted at 400 g for 5 min, and the pellets were washed three times with ice-cold PBS, and then resuspended in 500 μl of PBS. After addition of 10 μl of RNase (10 mg/ml), cells were stained with 10 μl of propidium iodide (1 mg/ml) at 37°C for 30 min in the dark. Flow cytometric analysis was performed using a Coulter Epics Elite flow cytometer (Beckman-Coulter, CA, USA).

Gametocytes were cultured and maintained using the PfDynGFP/PfP47mCherry reporter line as previously described [5 (link), 25 (link)], and were sorted using the Coulter Epics Elite flow cytometer (Beckman Coulter) or the BD FACS Aria SORP flow cytometer keeping cells at 4 °C in SA buffer at Radboud University (Nijmegen, Netherlands) [25 (link)]. The sorted female and male gametocytes (10⁶ gametocytes/mL) were stored in RNA Protect Cell Reagent and then frozen (Qiagen). Gametocytes were thawed and RNA extracted using methods described above. A tenfold dilution series of the extracted RNA was made ranging from 10⁶ to 10 female gametocytes/mL and 10⁵ to 1 male gametocytes/mL for calculation of the conversion factor for each assay. The male and female dilution series were run in duplicate on 3 separate occasions (a total of 36 PCR reactions per assay using aliquots of the same dilution series). The pfs25 and pfMGET mRNA transcript numbers were determined in these samples using the qRT-PCR assays with cRNA standards. These data sets are hereafter referred to as data set C (pfs25) and data set D (pfMGET). The study design was a randomized block design with the dilution series as treatments, daily runs as blocks and technical replicates used to estimate intra-assay variability.