Spleens were fixed in 4% methanol-free formaldehyde (Sigma, Cat. 28906) in PBS at 4 °C for 3.5 h followed by overnight incubation in 30% sucrose in PBS and freezing in OCT (Tissue-Tec, Cat. 4583). Spleens were cut into 10 µM sections using a Leica CM1850 cryostat and the tissue was transferred onto microscope slides. The tissue was blocked with PBS + 5% FCS + 2% rat serum + anti-FcR (clone 2.4G2, in-house hybridoma) for 1 h followed by staining with antibodies to Bcl6-CF568 (clone 7D1-10, in-house purified and conjugated to CF568 (Biotium, Cat. 92215); 1 in 200 dilution), CD35-biotin (BD Biosciences, Cat. 553816; 1 in 200 dilution), CD45.1-eFluor450 (clone A20, eBioscience, Cat. 48-0453-82; 1 in 200 dilution), CD45.2-FITC (clone 104, BD Biosciences, Cat. 553772; 1 in 200 dilution), and B220-AF647 (clone RA3-6B2, in-house purified and conjugated to Alexa fluor 647; 1 in 200 dilution) in a humidified chamber at 4 °C overnight. Slides were washed and stained with Streptavidin-Pacific orange (Thermo Fischer, Cat. S32365) at room temperature for 1 h. After washing twice for 1 h with PBS, slides were mounted using DAKO fluorescent mounting medium (DAKO, Cat. S3023) and Menzel X1000 Coverslip #1.5. Images were acquired on a Nikon AR1 confocal microscope with a CFI Plan Fluor ×20 MI lens using glycerol as immersion medium.
Streptavidin pacific orange
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin-Pacific Orange is a fluorescent labeling reagent. It consists of the streptavidin protein covalently attached to the Pacific Orange fluorescent dye. Streptavidin has a high affinity for the biotin molecule, making it useful for detecting and quantifying biotinylated targets.
Automatically generated - may contain errors
Lab products found in correlation
Ter119, by BioLegend (2 mentions)
Cd45.2 fitc, by BD (1 mentions)
B220 af647, by BD (1 mentions)
Cm1850 cryostat, by Leica (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Sca 1, by Thermo Fisher Scientific (1 mentions)
Sca 1, by BioLegend (1 mentions)
Cd150, by BioLegend (1 mentions)
Cd150, by Thermo Fisher Scientific (1 mentions)
Il 7rα, by BioLegend (1 mentions)
Cytobank, by Beckman Coulter (1 mentions)
Facsymphony a3, by BD (1 mentions)
Lsrii instrument, by BD (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
F4 80 apc, by Thermo Fisher Scientific (1 mentions)
Cd45 percp cy5, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Gr 1 rb6 8c5, by Thermo Fisher Scientific (1 mentions)
Cd45 apc cy7, by Thermo Fisher Scientific (1 mentions)
B220 ra3 6b2, by Thermo Fisher Scientific (1 mentions)
8 protocols using streptavidin pacific orange
1
Immunofluorescent Staining of Murine Spleen
2
Hematopoietic Stem Cell Immunophenotyping
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Bone marrow cells were ACK treated and then stained with antibodies Ter119 (BioLegend, 116204, 1/100), B220 (BioLegend, 103204, 1/100), Mac1 (BioLegend, 101204, 1/100), CD3 (BioLegend, 100244, 1/100), Gr1 (BioLegend, 108404, 1/100) (Lin), cKit, Sca1, CD34, Flk2, CD150, IL-7Rα FcγRα (eBioscience, 45-0161-82, 1/100), and PI (Supplementary Table 2 ). Biotinylated antibodies were stained using Streptavidin-Pacific Orange (Thermo Fisher Scientific) before staining PI. Posttransplant marrow was stained with antibodies Ter119, B220, Mac1, CD3, Gr1, IL-7Rα (Lin), cKit, Sca1, CD34, Flk2, CD150, FcγRα, CD45.1 (BioLegend, 110728, 1/100), CD45.2, and PI.
3
Comprehensive Hematopoietic Stem Cell Immunophenotyping
4
Quantification of SIgA and E. coli Complexes
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The amount of free and E. coli‐complexed SIgA in cell culture supernatants was determined by flow cytometry. Supernatants were harvested 3 h after addition of the stimuli (e.g. HS and SIgA) to the cultures and kept at −20°C until assayed. In these studies, uninfected cells (medium only) or cultures with IgG were used as controls. Briefly, supernatants were collected into 4·0 ml tubes, washed by centrifugation in PBS and stained with biotinylated mouse anti‐IgA1/IgA2 mAb (BD) and rabbit anti‐E. coli–IgG polyclonal antibody (Abcam). After an incubation of 45 min at 37°C, samples were washed twice with PBS and incubated for an additional 45 min at 37°C with streptavidin‐Pacific Orange (Invitrogen). After washing and fixation, specimens were analysed by flow cytometry on a custom LSR‐II instrument (BD Biosciences). Data were analysed with WinList version 7·0 (Verity Software House, Topsham, ME, USA).
5
Cardiac Myeloid Cell Profiling
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To characterize myeloid cell types in the heart after treatment with LNP4 formulation or control according to the protocol described earlier, single-cell suspensions from saline perfused hearts were minced and digested in DMEM with Collagenase I at 3 mg/mL for 45 minutes at 37°C. To deactivate the enzyme, the same amount of fetal bovine serum was added to each sample. Samples were filtered through 40 μm cell strainers. Red blood cell lysis was performed with ACK lysis buffer (Thermo Fisher Scientific). Samples were washed once with 200 µL of FACS buffer (PBS with 2% FBS and 2 mM EDTA) and resuspended in 100 μL of the same solution, and then incubated with a mix of 30 μL/well of antibodies CD45-PerCP-Cy5.5 (1:200, BioLegend, no. 109828), CD11b-SB600 (1:500, Invitrogen, no. 63–0112-82), F4/80-APC (1:200, Invitrogen, no. 17–4801-82), Gr1-Biotin (1:200, Biolegend, no. 108404), and Streptavidin-PacificOrange (1:500, Invitrogen no. S32365) at RT for 20 minutes in the dark . The cells were then analyzed using a BD LSRII flow cytometer (BD Biosciences). Neutrophils were gated as CD11b+Ly6Ghigh. Monocytes were gated as CD11b+Ly6ChighMHCIIlow. Macrophages were gated as CD11b+Ly6G−Ly6Clow F4/80+ cells.
6
Isolation and Characterization of Murine Hematopoietic Stem and Progenitor Cells
7
Cryopreserved PBMC Characterization and Analysis
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Cryopreserved PBMC were thawed and allowed to rest overnight (37°C, 5% CO2) as previously described [41 (link), 42 (link)]. Plating of cells (1x106), staining for viability, bacteria binding (50:1—bacteria:cells ratio [43 (link)]), blocking (human IgG -25 μl of a 1 mg/ml solution; mouse IgG -25 μl of a 200 μg/ml solution) and staining of surface targets with monoclonal antibodies were performed as described in detail in [40 (link)]. Monoclonal antibodies (mAbs) against the following molecules were used: CD19-ECD (clone J3-119; Beckman Coulter -BC-), CD38-PE-Cy5 (clone LS1298-4-3; BC), CD14-QDot 655 (clone TuK; Invitrogen), CD21-BV711 (clone B-ly4; Becton-Dickinson -BD-), integrin α4β7-Alexa647 (clone ACT-1; Millennium, The Takeda Oncology Co), CD3-Alexa Fluor 700 (clone UCHT1; BD), IgD-FITC (polyclonal goat anti-sera; Southern Biotech), CD27-PE (clone L128; BD), CD40-PE-Cy7 (clone 5C3; BD), IgA-Biotin (polyclonal goat anti-sera; Southern Biotech) and Pacific Orange-Streptavidin (Invitrogen, USA). Finally, stained cells were fixed with 1% PFA in PBS until data collection in a LSRII (BD) instrument.
8
Multiparameter Flow Cytometry of Immune Cells
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Cells were suspended and stained in FACS (fluorescence-activated cell sorting) buffer (2% newborn calf serum, 2 mM EDTA, and 0.1%NaN3 in PBS). For intracellular staining, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) following the manufacturer’s instruction. Antibody and reagents used for flow cytometry included the following: PE anti-T-bet (4B10), PE-CF594 anti-B220 (RA-6B2), APC-eF780 anti-B220 (RA3-6B2), PerCP-Cy5.5 anti-CD8 (53–6.7), PE-Cy7 anti-IgM (R6–60.2), FITC anti-IgM (R6–60.2), BV711 anti-IgD (11-26c.2a), PerCP-Cy5.5 anti-IgD (11-26c.2a), PE anti-GL-7 (GL-7), eF450 anti-GL-7 (GL-7), AF700 anti-CD38 (90), BV650 anti-CD19 (6D5), PE-Cy7 anti-CD19 (6D5), FITC anti-IgM (RMM-1), PE-Cy7 anti-IgM (1l/41), BV605 anti-CD73 (TY/11.8), PE anti-PD-L2 (TY25), FITC anti-CD45.1 (A20), Pacific Blue anti-CD45.1 (A20), APC-eF780 anti-CD45.2 (104), Pacific Blue anti-CD45.2 (104), PE-Cy7 streptavidin (BD Biosciences), and Pacific orange streptavidin (Invitrogen). All data were collected on a BD LSR Fortessa cytometer (BD) and analyzed with FlowJo (BD).
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