Treated and untreated cells were stained for viability using blue fluorescent reactive dye (Invitrogen). Cells were washed and blocked using human BD Fc block (BD Biosciences) in 3% BSA in PBS and stained with anti-CD11c-BV421 (clone: Bu15; Biolegend). In-house generated rabbit anti-human PAD4 antibody was fluorescently labelled using AF647 antibody labelling kit (Invitrogen) and used at 0.01 µg µl−1. Equal amounts of AF647-labelled rabbit IgG (3452S, Cell Signaling Technology) served as isotype control. Cells were incubated with labelled antibodies for 30 min at 4°C and flow cytometry was conducted at the Johns Hopkins Bayview Immunomics Core Facility using Cytek Aurora (Cytek Biosciences). Data were analysed using FCS Express 7 Research Edition (De Novo Software).
Blue fluorescent reactive dye
Manufactured by Thermo Fisher Scientific
The Blue fluorescent reactive dye is a laboratory product used for labeling and detection applications. It is designed to emit blue fluorescence when excited by the appropriate wavelength of light, enabling visualization and identification of target molecules or structures in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Af 647 antibody labelling kit, by Thermo Fisher Scientific (1 mentions)
Anti cd11c bv421, by BioLegend (1 mentions)
Human fc block, by BD (1 mentions)
Fcs express 7 research edition, by De Novo Software (1 mentions)
Tcrβ bv605, by BD (1 mentions)
Cd4 bv711, by BD (1 mentions)
Ptbk1 pe, by Cell Signaling Technology (1 mentions)
Anti cd45 af700, by BD (1 mentions)
F4 80 bv650, by BioLegend (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Ly6c apc cy7, by BioLegend (1 mentions)
Cd206 pe, by BioLegend (1 mentions)
Ly6g bv510, by BioLegend (1 mentions)
Anti cd11b bv421, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Cd64 apc, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd19 bv785, by BD (1 mentions)
Cd10 bv605, by Thermo Fisher Scientific (1 mentions)
Cd38 percp ef710, by Thermo Fisher Scientific (1 mentions)
5 protocols using blue fluorescent reactive dye
1
Multiparameter Flow Cytometry Assay
2
Multicolor Flow Cytometry Assay for Immune Profiling
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Tumor cell suspensions (4 × 106) prepared by mechanical and enzymatic dissociation16 (link) were stained in 96-wells round bottom plates with live/dead staining (Blue fluorescent reactive dye, #L34962 Invitrogen) during 20 min at room temperature. For flow-cytometry analysis and sorting, Fc receptors were blocked with anti-FcR (anti-CD16 and CD32, at 5 µg/ml, Biolegend #101339). After two washes in PBS 2% FCS, cells were stained with the following antibodies (all used at 1:100): anti-CD11b-BV421 (#562605), CD64-APC (#558539), CD11c-PeCy7 (#557401), TCRβ-BV605 (#562840) and CD4-BV711 (#563050) all purchased from BD Pharmingen; anti-CD45-AF700 (#103128), Ly6C-APCCy7 (#128025), Ly6G-BV510 (#127633), F4/80 BV650 (#123149), CD206-PE (#141706), IA/IE-BV785 (#107645) all purchased from Biolegend, and CD8-PerCPef710 (#46-0081-82) purchased from eBioscience. After washing, cells were fixed in 1% PFA, stored at 4 °C, and acquired the next day on LSR II or FORTESSA (BD Bioscience). For detection of phosphorylated proteins, cell suspensions were stimulated 3 h with DMXAA 250 µg/ml, fixed immediately in PFA 4%, permeabilized with frozen methanol 90%, stained overnight with 1:100 pTBK1-PE (#13498) antibodies (purchased from Cell signaling), then washed and further stained for multicolor flow cytometry.
3
Analyzing Donor T Cell Dynamics
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Spleen and pLNs were collected from recipient mice on days 7, 14, or 21 post T cell transfusion and flow cytometry analyses were performed to assess the depletion, proliferation, phenotype, and functional state of recipient and donor origin cells. CD45.1 and CD45.2 were used as congenic markers to distinguish between donor and recipient cells. Single cell suspensions were first stained with blue-fluorescent reactive dye (Invitrogen) to exclude dead cells from analysis, then for surface markers, and subsequently for intracellular or intranuclear molecules following fixation and permeabilization with the CytoFix/CytoPerm or Foxp3 Buffer Sets (BD Biosciences and eBioscience). Fluorescence dye-conjugated Abs for cell surface CD45.1 (A20), CD45.2 (104), CD3 (145-2C11), CD4 (GK1.5), CD8 (53–6.7), CD25 (7D4), CD19 (1D3), NK1.1 (PK136), CD11b (M1/70), and Foxp3 (MF23) were purchased from BD Biosciences, eBioscience, or BioLegend. Treg were defined as CD4+Foxp3+ or CD4+CD25+ cells as indicated in figure legends, and CD4-Th cells were defined as CD4+Foxp3−. Data were acquired using a Canto II flow cytometer and analyzed using the FCS Express V4 software.
4
Multiparameter Flow Cytometry of B Cell Subsets
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All flow cytometry used appropriate isotype controls. Gating and compensation were aided by the performing fluorescence minus one controls. Cells isolated from biopsies or blood were stained with blue-fluorescent reactive dye (Life Technologies), CD19-BV785, CD27-APC (BD Biosciences) or PE or BV421, CD10-BV605 or APC, CD38-PerCp-ef710 (eBioscience), CD24-PE/Cy7 or BV605, IgD-APC/Cy7, IgM-V450 (BD Biosciences), IgG-PE/Cy7 and IgA-FITC (Miltenyi Biotec), in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before analysis on the BD LSRFortessa (BD Biosciences). For high-throughput sequencing analysis, cells were stained with LIVE/DEAD Fixable Aqua (Life Technologies) or DAPI, CD19-BV785, CD27-FITC or APC, IgD-APC/Cy7, CD38-PerCp-ef710 (eBiosciences), CD10-BV605 in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before sorted on the BD FACSAria (BD Biosciences). Four subsets from PBMC and three subsets from paired biopsy mononuclear cells from Donors 1 to 4. The numbers of cells used to generate sequences for each sample from Donors 1 to 4 are shown in Supplementary Fig. 14d .
5
Antigen Processing Assay for Dendritic Cells
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We performed a natural antigen processing assay (NAPA) as described previously (13 (link)). Briefly, immature mo-DCs were seeded in 6-well plates at a density of 1 × 106 cells in 1 mL of RPMI 1640 and 10% fetal bovine serum, supplemented with 2 mM L-glutamine and 20 μM ß-mercaptoethanol in duplicate. Immature mo-DCs were left at rest or stimulated with 1 μg/mL purified lipoteichoic acid (LTA) from Staphylococcus aureus (InvivoGen, San Diego, CA) or live B. burgdorferi strains A3 or B31 at multiplicities of infection of 1 or 10 bacteria per cell for 8 and 24 h. Cells were incubated at 37°C, 5% CO2, and >95% humidity. At the end of the incubation period, all samples were harvested into a microcentrifuge tube, pelleted at 1,200 rpm at room temperature for 3 min. Cells were washed with PBS/EDTA (PE) Buffer and pelleted at 1,200 rpm at room temperature for 3 min. Cells were stained with CD14-PE (BD Pharmingen), CD11c-PerCP (BioLegend), HLA-DR-BV510 (BD Horizon), and Blue Fluorescent Reactive Dye (Life Technologies) for 15 min in BV Buffer (BD Horizon). At the end of the incubation period, cells were washed twice in PBS and resuspended in 100 μL PBS for flow cytometry analysis in a BD FACSAria II flow cytometer (BD Biosciences). All data was gated on CD14−/ CD11c+ monocyte derived dendritic cells.
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