Lsrii facsfortessa

PrimeFlow RNA assay (Thermo Fisher Scientific, San Diego, CA) was performed on IHLs. The specific target probe set for mouse IL-36γ (Assay ID # VB1–3046123-PF, type 1) was designed by and purchased from Thermo Fisher Scientific. When applicable, cell surface staining was performed for 30 min at 4°C with fluorochrome-conjugated antibodies. Cells were then fixed for 30 min at 4°C. After permeabilization, cells were fixed a second time for 1 h at RT with Fixation buffer 2. A hybridization step was performed by incubating cells with the appropriate target probe sets for 2 h at 40°C. Samples were stored over night at 4°C in dark. Next day, pre-amplification and amplification of the hybridization were performed by two consecutive incubations of 1.5 h at 40°C with the pre-Amplification mix and subsequently the Amplification mix. Finally, cells were incubated with the label probe sets for 1 h at 40°C. Samples were processed on a LSRII FACS Fortessa (Becton Dickinson, San Jose, CA, USA), and data were analyzed using FlowJo X software (Tree Star, Ashland, OR).

Intracellular staining was performed according to our previous methods (35 (link)). Briefly, for detection of IFN-γ, TNF-α and IL-2, cells were incubated for 4 h with PMA (50 ng/ml) and ionomycin (750 ng/ml) in the presence of GolgiStop (BD Bioscience). For IL-17 and IL-22 detection, cells were cultured with rIL-23 (20 ng/ml) for 16 h. GolgiStop was added at the last 4 h of culture. The inhibitors of the mTORC1/PI3K pathway were added in selected experiments. After incubation, cells were collected, stained with fixable viability dye, blocked with FcγR blocker (CD16/32), and stained for specific surface molecules. After surface staining, cells were fixed, permeabilized and stained for intracellular cytokines by using a fixation/permeabilization kit (eBioscience). Samples were processed on an LSRII FACSFortessa (Becton Dickinson, San Jose, CA) and analyzed by using FlowJo software (TreeStar, Ashland, OR).

Flow cytometry staining was performed according to our previous methods (Jie et al., 2017 (link)). Briefly, isolated mononuclear cells from mice placenta were stained with fixable viability dye and purified anti-CD16/32 (clone 2.4G2) followed by surface staining of FITC-CD11b, APC-Ly6G, APC-CD3, Pacific blue-CD4, APC-780-CD8, PE-Cy7-CD122, FITC-CD45.1, and Percp-Cy5.5- CD45.2 (eBioscience). Samples were collected using LSRII FACSFortessa (Becton Dickinson) and analyzed using FlowJo software (Tree Star).

Leishmania amastigotes were labeled with 1 µM carboxyfluorescein succinimidyl ester (CFSE) in room temperature PBS for 15 min, followed by quenching in complete media and 2 additional washes in PBS. CFSE-labeled parasites were co-cultured with neutrophils at a multiplicity of infection (MOI) of 5 for 4 h, as described previously [7 (link)]. Cells were washed, blocked with anti-CD16/CD32, and stained with anti-Ly6G-AlexaFluor 647 (BioLegend, San Diego, CA) and anti-CD11b-PE-Cy7 (BD Bioscience, San Jose, CA). Cells were analyzed by using a LSRII FACSFortessa (BD Bioscience). Neutrophils were identified based on forward/side scatter characteristics and Ly6G/CD11b positivity, and cell infection was identified by neutrophil acquisition of CFSE. Parasite uptake was further characterized by using an ImageStreamx Mark II Imaging Flow Cytometer (Amnis Corporation, Seattle, WA) to count the number of parasites internalized by neutrophils. Images obtained from the imaging flow cytometer were captured at 60× magnification.

For surface staining, cells were first incubated with Fc Receptor Blocker (CD16/32), followed by fluorochrome-labelled Abs of surface markers. For intracellular cytokine staining, Brefeldin A (Biolegend) was added for last 6 h of culture. For analysis of virus-specific CD4 and CD8 T-cell responses, cells were incubated with viral peptides GP33 (5 μg/ml) and GP61 (5 μg/ml) for 5 h in the presence of Brefeldin A. After incubation, cells were stained for surface markers first at 4°C for 30 min in the dark, followed by intracellular staining using IC Fixation Buffer (Thermo Fisher Scientific). For Ki-67 staining, the Foxp3/Transcription Factor Staining Buffer Set was used. The phosflow experiments were performed according to the protocol of BD Biosciences. Briefly, cells were stimulated with cytokines for the indicated times, followed by immediate fixation using a pre-warmed Cytofix Fixation Buffer at 37°C for 12 min. The cells were permeabilized using chilled Perm Buffer III for 1 h on ice and then were washed and stained with phosflow Abs. To measure the mitochondrial membrane potential, TMRM assay kit was used (Abcam). Samples were processed on an LSRII FACS Fortessa (BD Bioscience, San Jose, CA) and analysed using FlowJo X software (Tree Star, Ashland, OR).