Facs canto flow cytometer

Cells were fixed with 4% PFA for 30 minutes and incubated in 1% Triton-X-100 for 15 minutes to permeate the cell membrane. Nonspecific binding was blocked with 1% BSA at room temperature for 1 hour. Proteins were detected with specific primary antibodies at 4 °C overnight. Primary antibodies were as follows: anti-NESTIN (1:100, MAB353; Millipore), anti-TuJ 1 (1:200, T2200; Sigma-Aldrich), anti-OCT4 (1:200, sc-8628; Santa Cruz), anti-NANOG (1:200, ab80892; Abcam), anti-phospho (Ser465/467)-SMAD2 (1:200, #3108; CST), and PAX6 (1:200, AB_528427; DSHB). After three washes with PBS, cells were incubated with corresponding secondary antibodies (1:1000; Jackson ImmunoResearch) for 1 hour. DNA was counterstained with Hoechst33342 (Invitrogen) for 5 minutes at room temperature. Immunofluorescent images were obtained on an Axioplan Zeiss microscope (LSM 780; Carl Zeiss). Quantitative analysis of immunofluorescent staining was performed using ImageJ software when the immunofluorescent images were obtained at the same exposure parameters.
For FACS analysis, cells were digested into single cells, followed by two washes in DPBS. The cells were then filtered through a 35-μm cell strainer cap (Falcon™ Cell Strainers, 352235). Sox1-GFP cells were sorted and counted by flow cytometry. Analysis was performed on a FACS-Canto flow cytometer (Beckman Coulter MoFlo™ XDP).

The frozen trophozoites were thawed as previously described (38 (link)). A 10% suspension of the thawed infected red blood cells pellet was prepared using 1XPBS. 2.5µL of the 10% pellet suspension was added into 8.5µL of 1XPBS/0.5%BSA then stained with 10µg/µL Ethidium Bromide (EtBr). The suspension was incubated in a U-bottomed 96 well plate with 1µL of test plasma and malaria naïve control plasma for 30 minutes at room temperature to allow for antigen-antibody binding. The cells were then washed three times using 1XPBS/0.5%BSA by centrifugation at 110×g for 3 minutes. 50µL of fluorescein isothiocyanate (FITC) conjugated sheep anti-human IgG (Binding Site, UK) was added at 1:50 dilution and this was incubated for 30 minutes in the dark, then washed 3 times with 1XPBS/0.5%BSA by centrifugation at 100×g for 3 minutes. The pellet was then suspended in 200µL of 1XPBS. 100µL of the re-suspended pellet was further diluted with 400µL of 1XPBS in a FACS tube to bring the pellet volume to 0.05µL and 1000 trophozoite-infected erythrocytes were acquired from each tube on a FACSCanto flow cytometer (Beckman Coulter, UK).