Acuri c6

Cell labeling of lung and blood leukocytes with monoclonal antibodies was performed as previously described [10 (link),35 (link)]. Separated lung cells or blood leukocytes (1 × 10⁶/well in 100 µL staining buffer) were incubated for 20 min at 4 °C with the following combinations of unlabeled primary monoclonal antibodies to cell surface antigens of camel leukocytes (mAbs; Table 1): CD45/CD172a, CD163/MHC-class II, or CD4/WC1 or with a combination of anti-CD11a-PE and anti-CD18-FITC mAbs. After two washing steps (by adding 150 µL washing buffer followed by centrifugation at 300× g for 3 min), the cells were stained with fluorochrome-labeled antibodies to mouse IgG1, IgG2a, and IgM isotypes for 20 min at 4 °C. In a third staining step, PerCP-labeled monoclonal antibodies to CD14 were added to the cells stained with CD45 and CD172a. Staining with mouse isotype control antibodies was also performed. After the final cell wash, the Acuri C6 flow cytometer (Accuri C6, Becton Dickinson Biosciences) was used for the acquisition of at least 100,000 total cells. CFlow Software, Version 1.0.264.21 (BD Biosciences) was used for data analysis.

Apoptotic response to treatment was quantified using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI; eBioscience). All cell lines were treated with either single agent or combination drugs for 24 hours in 0.1% FBS media prior to staining with annexin V-FITC (1:400) for 5 minutes and PI for 10 minutes at room temperature (RT). Cells were treated with 100 nM doxorubicin for 24 hours as a positive control for apoptosis. Collected conditioned media and cell fractions were run on a BD Acuri C6 flow cytometer (BD Biosciences) with a threshold of 5 x 10⁴ events per group. Laser intensities were based upon control readings and each data set was compensated to account for spillover between FITC and PI (FL1 and FL3 respectively). Early apoptosis was defined as annexin V positive and PI negative, while late apoptosis was defined as annexin V positive and PI positive.

To perform cell phenotype analysis, single cell suspensions from the spleen were isolated from homogenized tissues. For cell phenotype analysis, 1 × 10⁶ splenocytes were stained with surface marker antibodies including CD3e-PE-Cy5, CD4-FITC, CD8a-PE (BD Biosciences, Franklin Lakes, USA). Stained cells were acquired on a BD FACSCalibur and BD AcuriC6 and analyzed using Flow Jo software and BD AcuriC6 software.

Single cell suspensions of neurospheres were stained with Carboxyfluorescein succinimidyl ester (CFSE) using CellTrace CFSE Cell Proliferation Kit (Life technologies, Oregon, USA cat# C34554) following manufacturer’s protocol. The stained cells were divided into two equal parts, one part treated with DMSO and the other part was treated with 50μM vismodegib for 5 days. CFSE staining intensities were measured by flow cytometry (BD Acuri C6, BD BioSciences, USA) after 5 days of treatment.

To perform cell phenotype analysis, single cell suspensions from lung and spleen were isolated from homogenized tissues. 1 × 10⁶ splenocytes and lung leukocytes were stained with surface marker antibodies including CD45-PerCP, CD11b-APC, CD23-FITC, F4/80-PE, CD11c-FITC, SiglecF-PE, B220-FITC, CD27-PE-Cy7 and IgG1-PE (BD Biosciences). Stained cells were acquired on a BD FACSCalibur and BD AcuriC6 and analyzed using Flow Jo software and BD AcuriC6 software.

Following 48-hour treatments, cells were pelleted by centrifugation at 1500 rpm for 5 minutes using the Sigma 1–15 benchtop centrifuge. Positive control and negative control samples were FCCP (Sigma-C2920)—and 95% ethanol-treated samples respectively. The supernatant was discarded, and the pellet resuspended gently in 400 μl of JC-10 dye-loading solution (MAK160-1KT). The cells were incubated at 37°C, 5% CO₂ and 95% humidity for 60 minutes. Fluorescence intensity was monitored using FL1 (green) and FL2 (orange) fluorescent channels in a BD Acuri C6 flow cytometer (BD Biosciences, CA, and USA). For proper gating, an untreated unstained sample was included to determine the location of non-fluorescent cells in the quadrants and the gates for all other samples were set accordingly (S1 Fig). Fluorochrome spill over between FL1 and FL2 channels was corrected by fluorescence compensation. FL1 was corrected by subtracting 3.2 units from FL2, while FL2 was corrected by subtracting 7.5 from FL1. All samples were run in triplicates. FCCP dissolved in 95% ethanol at 10 μM was added as a positive control. A vehicle-only control (cells treated with 95% ethanol only) was also included.