S1000 flow cytometer

Synchronized cells were trypsinized and harvested, and after one PBS wash, cells were fixed in 70% cold ethanol for 30 min at 4°C or overnight at −20°C. After one PBS wash, the fixed cells were incubated with staining buffer (20 μg/mL RNase A (Thermo Fisher, EN0531) and 50 μg/mL Propidium Iodide (PI) in PBS (G Biosciences, 786–1273)) at 37°C for 30 min, followed by one PBS washing, and resuspended in 300 μL PBS. The cells were passed through a 40-μm Nylon Mesh (Fisherbrand, 22363547) before analysis with an S1000 Flow Cytometer from Stratedigm. At least 10,000 cells were counted and PI signal quantified using FlowJo software (version 10.8.1).

Blood samples were taken from mice by retro-orbital bleeding and centrifuged at 1150g for 5 min at room temperature to separate plasma from the cell pellets. Plasma was removed and frozen at −80°C for subsequent analysis. The cell pellets were resuspended in 1.1 ml of 1× RBC lysis buffer (BioLegend) and incubated on ice for 10 min. After RBC lysis, each sample was pelleted at 1150g in a centrifuge for 5 min at room temperature and then stained with 50 μl of an antibody cocktail containing 1:100 diluted anti-human CD3-FITC (BioLegend, clone UCHT1), 1:100 diluted anti-human CD4-PE (BioLegend, clone RPA-T4), and 1:100 diluted anti-human CD8-APC (BioLegend, clone RPA-T8) in PBS+ for 30 min on ice. Samples were then analyzed on a Stratedigm S1000 flow cytometer. Samples were first gated by CD3 expression before determining the ratio of CD4 to CD8 within this subset. Samples containing fewer than 20 CD3⁺ events were excluded from the analysis.

Total RNA was extracted from targeted tissues using either the RNeasy Mini Kit (QIAGEN, CA) or TRIzol Reagent (Thermo Fisher Scientific, MA) following the manufacturer’s protocols. cDNA was synthesized using the iScript RT-qPCR kit (Bio-Rad, CA). Quantitative PCR was performed by using iTaq SYBR Green Supermix (Bio-Rad, CA) on a CFX96 real time system (Bio-Rad). PCR involved an initial denaturation at 95°C for 5 min, 45 cycles of 10 sec at 94°C, 10 sec at 58°C, and 10 sec at 72°C. Fluorescence readings were taken at 72°C after each cycle. At the end of each reaction, a melting curve (70 - 95°C) was checked to confirm the identity of the PCR product. Relative expression of AgTRIO was calculated by normalization to A. gambiae actin mRNA. The Plasmodium load in mice was determined by PCR using primers to amplify P. berghei 18S rRNA and normalized to M. musculus beta-2 microglobulin (Table S2). Parasitemia levels were also quantified by flow cytometric analysis based on the fact that parasites carry GFP. Flow cytometric analysis allows distinction between infected mice and healthy controls, and was confirmed by thin blood smears. S1000 flow cytometer (Stratedigm, CA) was used for data acquisition. Flow cytometry data were analyzed with FlowJo software (Ashland, OR).