Live dead fixable violet dead cell stain

Flow cytometry was performed on the dissociated colon cell suspension using a BD LSR Fortessa (BD Bioscience) and analyzed with Diva Software. Viable cells were negative for the staining with Live/Dead Fixable Violet dead cell stain (Lifetechnology). For the evaluation of immune populations, cells were then labeled with the following antibodies: CD45-PerCPVio700, CD11b-FITC, F4/80-APC, CD3-PE, CD8-VioGreen, CD4-VioBrightFITC, and CD25-PEVio770 (Miltenyi Biotec).
T lymphocyte profiling studies were performed by intracellular staining with the inside Stain Kit and the following antibodies: IFN-γ-FITC, IL-2-PE, and TNF-α-APC for the Th1 profile and IL-10-FITC, IL-4-PE, and IL-5-APC for the Th2 profile and Treg were evaluated with Foxp3-Vio667 as recommended by the manufacturer (Miltenyi Biotec).
Appropriate fluorochrome-matched isotype antibodies were used to determine nonspecific background staining. All stainings were performed on 100 µl of PBS without calcium and magnesium and 1% heat-inactivated FCS.
A multiplex bead-based immunoassay was used (Biolegend, LEGENDPlex™ Mix and Match System) for cytokine and Arginase-1 titration from IBD patients’ monocytes.

Human primary astrocytes (HA) derived from brain cerebral cortex were purchased from ScienCell Research Laboratory and were cultured according to the provider’s instructions. Briefly, HA were grown and cultured at 37 °C with 5 % CO₂, in astrocyte medium (AM), supplemented with 2 % fetal calf serum (FCS) and 1 % astrocyte nutritive supplement with 1 % penicillin/streptomycin (referred as “complete astrocyte medium”). For immunofluorescence, cells were fixed for 15 min with 4 % paraformaldehyde and stored in phosphate-buffered saline (PBS) at 4 °C. For flow cytometry, cells were resuspended with trypsin (BioConcept) and first labeled with LIVE/DEAD fixable violet dead cell stain (Life Technologies). Then, HA were stained with cytofix/cytoperm (BD Biosciences) with mouse anti-IL-22R1 (clone 305405, R&D Systems)/mouse IgG₁ isotype control (clone 11711, R&D Systems) or rabbit anti-IL-10R2 (sc-69580, Santa Cruz Biotechnology)/rabbit IgG isotype control (AB-105-C, R&D Systems) primary antibodies and followed by donkey anti-mouse IgG AF546 and goat anti-rabbit IgG AF488 (Invitrogen) secondary antibodies. Alternatively, cell suspension was directly used for staining as described in “Proliferation and cell death/apoptosis assays” section. Cells were processed with an LSRII flow cytometer (BD Biosciences) and were analyzed with FlowJo software (version 9.1.11, Treestar).

At day 28 iMGLs cultures were stained with 100nM LysoTracker-Red (Thermo Fisher Scientific, L7525) for 30min at 37°C followed by 1μM LysoSensor-Green (Thermo Fisher Scientific, L7535) staining for 1min at 37°C. To characterize hydrolytic capacity of lysosomes, cells were stained with 1μg/ml DQ Red BSA (Thermo Fisher Scientific, D12051) for 1h at 37°C. Cells were also stained with LIVE/DEAD Fixable Violet Dead Cell Stain (Life Technologies, L34973) to exclude dead cells. After collecting the cells, single cell data were acquired using Attune flow cytometer (Thermo Fisher Scientific) and analyzed using FCS Express 7 (De Novo Software). Gates were set up based on fluorescence minus one (FMO) controls. In addition, we quantified DQ-BSA red fluorescent signal over time using the Incucyte S3 live imaging system. Cells were plated in 96-well plates (40,000 cells/well) and treated with 1μg/ml of DQ Red BSA. Images were acquired every 30min over 5h at 37°C. Total integrated density was calculated as mean red fluorescent intensity multiplied by surface area of masked object (i.e. cell), [RCU × μm²].

To assess the effect of enzalutamide on the cell surface phenotype of breast cancer cells, ZR75-1, BT549 and MDA MB 231 cells were treated with either enzalutamide or vehicle for 48 hours. After 48 hours, cells were harvested and stained with the following antibodies: HLA A2-PE-Cy7 (MHC-I), MUC-1-FITC (TAA), CD54-BV421 (ICAM-1), CD95-FITC (Fas) (BD Biosciences, San Jose, CA), CEA-APC (TAA) (Miltenyi Biotec, Auburn, CA), TRAIL receptor 1 and TRAIL receptor 2 (R & D Systems, Minneapolis, MN). LIVE/DEAD Fixable Violet Dead Cell Stain (Life Technologies, Grand Island, NY) was used to determine cell viability. Cells were incubated with the antibodies for 30 min at 4°C, acquired on a FACS Verse flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and analyzed using FlowJo software (TreeStar, Inc., Ashland, OR).

Single-cell suspensions were prepared by gentle mechanical disruption of spleens, followed by lysis with ACK lysis buffer to remove RBCs, then surface-stained with anti-CD4 (RM4-5), CD8 (53–6.7), CD45.1 (A20), CD44 (IM7), PD-1 (RMP1-30), B220 (RA3-6B2), CD19 (ebio1D3), CD95 (Jo2), or T cell– and B cell–activation antigen (GL7) antibodies (directly conjugated by FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, BV421, or BV605, respectively). Dead cells were excluded by using LIVE/DEAD Fixable Violet Dead Cell Stain (Life Technologies). A three-step CXCR5 staining was performed as previously described using purified rat anti-mouse CXCR5 (BD), a secondary biotin-conjugated Affinipure Goat anti-rat IgG (Jackson ImmunoResearch), and finally with streptavidin-APC (Invitrogen) or streptavidin-BV605 (Bioclone)40 (link). For Bcl6 (K112-91) staining, the cells were stained for surface antigens, followed by permeabilization, fixation, and staining using the Foxp3 Permeabilization/Fixation Kit and Protocol (eBioscience). Intracellular cytokine staining of IFN-γ (XMG1.2), TNF-α (MP6-XT22), and IL-2 (JES6-5H4) was performed using the Cytofix/Cytoperm Kit (BD Pharmingen) according to the manufacturer’s instruction. All of the staining antibodies were purchased from BD, eBioscience, or BioLegend. Flow cytometric data were collected on a FACS Canto II (BD) and analyzed using the FlowJo software (TreeStar).

The ALDEFLUOR kit (StemCell Technologies Vancouver, Canada) was used according to manufacturer’s protocol. Cells were sorted by using a FACS Aria II (BD Biosciences, San Jose, CA) with 130-μm nozzle. Cell viability was assessed with LIVE/DEAD Fixable Violet Dead Cell Stain (Life Technologies, Carlsbad, CA). Sorted cells were cytospun onto glass slides for immunofluorescent analysis.