Lsrii green

BALFs were collected by making a longitudinal incision on the ventral side of the neck exposing the trachea and lavaging the lung twice with 0.8 ml PBS, pH 7.4. Total leukocyte counts were determined using a hemocytometer.
For flow cytometric analysis, BAL cells were incubated with 2.4G2 mAb against FcγRII/III, and stained with APC-conjugated anti-CD11c (Caltag Laboratories), BUV395-, PE-, or FITC-conjugated anti-CD11b (BD Biosciences), FITC-, PE- or PE-Cy7-conjugated anti-Ly6G mAb (Clone 1A8, BD Biosciences), PerCP-Cy5.5-conjugated anti-Ly6C mAb (Biolegend), and BV421-conjugated anti-Siglec-F (BD Biosciences). The stained cells were analyzed on a BD LSRII-green using BD FACSDiva and FlowJo software analysis.

Cell surface IFNαR2 levels were quantified in HCV-infected RLW cells using directly conjugated anti-IFNαR2-Pycoerythrin (PE) antibody. Fluorescence was detected by flow cytometry (BD LSR-II-Green).

Immature D16 hiPSC-astrocytes were plated on 10 cm dishes with a density of 500,000/dish. On the day of the experiment, immature D20 astrocytes were collected using TrypLE^™ Express (Gibco, 12605-028) and prepared as a single cell suspension (1×10⁷ cells/mL) with flow cytometry staining buffer (10 % bovine serum albumin (BSA) in PBS with 0.1 % sodium azide). The cell suspension was aliquoted to 100 μL per tube (Falcon, 352054) and incubated with a LIVE-DEAD^™ Fixable Blue Dead Cell Stain Kit (Invitrogen, L34961) and 1 μL of mouse anti-CD44 antibody (Abcam, ab6124) per tube for 30 minutes on ice. Cells were washed with PBS by centrifuging at 500 × g for 5 minutes at 4°C. Cell pellets were resuspended in 100 μL of staining buffer containing 1 μL of goat anti-mouse Alexa Fluor 594 secondary antibody (Thermo Fisher Scientific, A-11005) and incubated for 30 minutes at RT, followed by three washes with PBS by centrifuging at 500 × g for 5 minutes at 4°C. Cells were resuspended in 1 % BSA in PBS before running flow cytometry. The flow cytometry BD LSR II Green (BD Biosciences) was used to detect CD44 labeling at 605 nm excitation wavelength and cell viability at 450 nm excitation wavelength. Forward scatter, side scatter and doublet discrimination were applied to screen for single astrocytes and assess the percentage of live and dead cells in the sample.