Pe cy7 anti mouse cd19

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PE/Cy7 anti-mouse CD19 is a fluorescently-labeled antibody that binds to the CD19 protein expressed on mouse B cells. It can be used for the identification and enumeration of B cells in flow cytometry applications.

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Anti-CD19 (91 citations) CD19-PE (55 citations) CD19 PE-Cy7 (36 citations) Anti-CD19-PE-Cy7 (26 citations) Anti-CD19-PE (22 citations) PE anti-mouse CD19 (4 citations)

8 protocols using pe cy7 anti mouse cd19

Pulmonary Immune Cell Phenotyping

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At 7 dpi, pulmonary single-cell suspension was obtained and labeled using the method described above but with the following labeling antibodies: APC anti-mouse CD11c, FITC anti-mouse MHC Class II, PE anti-mouse NKp46, PE/Cy7 anti-mouse CD19, PerCP/Cy5.5 anti-mouse CD3ε, PE anti-mouse F4/80, PE/Cy7 anti-mouse Ly-6G, PerCP/Cy5.5 anti-mouse Siglec H (all from BioLegend, USA), FITC anti-mouse CD4 and PE anti-mouse CD8a (both from eBioscience).

Liao Y., Liu X., Huang Y., Huang H., Lu Y., Zhang Y., Shu S, & Fang F. (2017). Expression pattern of CD11c on lung immune cells after disseminated murine cytomegalovirus infection. Virology Journal, 14, 132.

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Flow Cytometry Analysis of Antigen-Specific T Cells

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Flow cytometry analysis was performed using a BD Accuri 6 plus (BD Biosciences) and analyzed by FlowJo software (Tree Star, Ashland, OR, USA). Epitope- specific T cells were studied using MHC Class I Pentamers (F093-84C-E, ProImmune, Oxford, UK). Other antibodies used included the following: murine Fc block CD16/32 (101320, Biolegend); FITC anti-mouse CD8 (A5402-3bE, ProImmune); PE/Cy7 anti-mouse CD19 (115520, Biolegend), FITC anti-mouse CD11c (117306, Biolegend), APC anti-mouse H-2Kb bound to SIINFEKL(116619, Biolegend), PE anti-mouse CD370 (143504, Biolegend), PE anti-mouse PD-1 (135206, Biolegend), PE anti-mouse CD68 (137013, Biolegend), APC anti-mouse CD4 (100412, Biolegend). All staining procedures were performed according to the manufacturer’s recommendations. Gating strategies are shown in supplementary Fig. 12.

Fusciello M., Fontana F., Tähtinen S., Capasso C., Feola S., Martins B., Chiaro J., Peltonen K., Ylösmäki L., Ylösmäki E., Hamdan F., Kari O.K., Ndika J., Alenius H., Urtti A., Hirvonen J.T., Santos H.A, & Cerullo V. (2019). Artificially cloaked viral nanovaccine for cancer immunotherapy. Nature Communications, 10, 5747.

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Multiparametric Immune Cell Analysis

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The following antibodies were used in the experiments: TruStain Fc block anti-mouse and anti-human CD16/32 (101320, BioLegend), FITC anti-mouse CD8 (A502-3B-E, ProImmune), phycoerythrin-cyanin 7 (PE/Cy7) anti-mouse CD3e (100320, BioLegend), PE/Cy7 anti-mouse CD19 (115520, BioLegend), FITC anti-mouse CD11c (553801, BD Bioscience), APC anti-mouse H-2K^b bound to SIINFEKL (141606, BioLegend), APC mouse IgG (usually mouse IgG) κ isotype Ctrl (400119, BioLegend). SIINFEKL epitope-specific T cells were studied using R-PE-labeled H-2K^b/SIINFEKL pentamer (F093-84A-E, ProImmune). Trp2_(180–188) epitope-specific T cells were studied using R-PE-labeled H-2K^b/SVYDFFVWL pentamer (F185-2B-E, ProImmune), and gp100_(25–33) epitope-specific T cells were studied using APC-labeled H-2K^b/KVPRNQDWL pentamer (F1333-4B-E, ProImmune).
Flow cytometric analyses were performed using a Gallios flow cytometer (Beckman Coulter) or BD Accuri 6C plus flow cytometer (BD Biosciences). FlowJo software v10 (FlowJo) was used for the data analysis.

Ylösmäki E., Malorzo C., Capasso C., Honkasalo O., Fusciello M., Martins B., Ylösmäki L., Louna A., Feola S., Paavilainen H., Peltonen K., Hukkanen V., Viitala T, & Cerullo V. (2018). Personalized Cancer Vaccine Platform for Clinically Relevant Oncolytic Enveloped Viruses. Molecular Therapy, 26(9), 2315-2325.

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Multiparametric Immune Cell Analysis

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Blood was centrifuged at 400 x g for 15 minutes. The blood pellet was repeatedly incubated in red blood cell lysis buffer (4.15 g NH₄Cl, 0.55 g KHCO₃, and 0.185 g EDTA disodium salt in 500 ml H₂O) centrifuged until a white pellet was obtained. After adjusting the samples to 1 million cells per 100 μl PBS, the Fc receptors of immune cells were blocked with 10 μg/ml unlabeled purified rat anti-mouse CD16/CD32 antibody (#553142, BD Biosciences, Germany). Dead cells were stained with the Zombie Yellow™ Fixable Viability Kit (#423103, BioLegend, US). After centrifuging at 400 x g for 3 min, cells were incubated with 100 μl of an antibody mix containing FITC anti-mouse CD3 (#100203, BioLegend, US), APC anti-mouse CD8a (#100711, BioLegend, US), Pacific Blue anti-mouse CD4 (#100427, BioLegend, US), PE/Cy7 anti-mouse CD19 (#115519, BioLegend, US), APC/Cy7 anti-mouse NK-1.1 (#108723, BioLegend, US), Alexa Fluor700 anti-mouse/human CD11b (#101222, BioLegend, US), and a PE anti-mouse/rat/human CD27 (#124209, BioLegend, US) antibody. Unstained samples were used to control for autofluorescence during the flow cytometry measurements. Cells were measured by flow cytometry (BD LSR II FortessaT M SORP, BD Bioscience, Germany). The data were acquired with the BD FACSDiva™ Software and analyzed with FlowJo V10.5. The gating strategy is described in Figure S4B.

Leimbacher A.C., Villiger P., Desboeufs N., Aboouf M.A., Nanni M., Armbruster J., Ademi H., Flüchter P., Ruetten M., Gantenbein F., Haider T.J., Gassmann M, & Thiersch M. (2023). Voluntary exercise does not always suppress lung cancer progression. iScience, 26(8), 107298.

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Generating FITC and PE Labeled MD39 Tetramers

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To generate FITC and PE labelled MD39 tetramers, biotinylated avi-tagged MD39 produced as previously mentioned were incubated with molar ratios of Streptavidin-FITC or Streptavidin-PE (BioLegend) for 30 min at room temperature^{8 (link),56}. Single-cell suspensions from spleen or lymph nodes of the animals were first washed with PBS, then stained with live dead dye (fluorescent violet reactive, Thermo Fisher) diluted in PBS at room temperature for 10 min, followed by the addition of mouse Fc-block (anti-mouse CD16/32, BioLegend) diluted 1:100 in 1% FBS/PBS and incubation at room temperature for additional 30 min. The cells were washed, and then incubated with 5ug/mL Streptavidin-MD39-FITC and Streptavidin-MD39-PE at room temperature for 45 min. Antibody mixtures (anti-mouse CD19-PE-Cy7, Anti-mouse IgD APC-Cy7, and Anti-mouse IgM BV711, BioLegend, each diluted 1:200 in 1% FBS/PBS) were then added to the cells without any intermediate washing steps, followed by incubation for another 45 min at room temperature. The cells were washed once with 1% FBS/PBS, and then resuspended at 10 million/mL concentration in 1% FBS/PBS for sorting with the FACS ARIA II instrument. CD19 + IgM− IgD− MD39-Tetramer-FITC + MD39-Tetramer-PE + cells were then sorted in bulk into 1.5 mL Eppendorf tube containing 1% FBS/PBS for downstream cDNA prep with 10× genomics.

Xu Z., Walker S., Wise M.C., Chokkalingam N., Purwar M., Moore A., Tello-Ruiz E., Wu Y., Majumdar S., Konrath K.M., Kulkarni A., Tursi N.J., Zaidi F.I., Reuschel E.L., Patel I., Obeirne A., Du J., Schultheis K., Gites L., Smith T., Mendoza J., Broderick K.E., Humeau L., Pallesen J., Weiner D.B, & Kulp D.W. (2022). Induction of tier-2 neutralizing antibodies in mice with a DNA-encoded HIV envelope native like trimer. Nature Communications, 13, 695.

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Lymphocyte Profiling in Vaccinated Mice

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Lymph nodes and spleens were harvested from vaccinated mice. These organs were then processed into a single cell suspension after being passed through a 40 μM strainer, lysed with ammonium-chloride-potassium (ACK) solution, and washed in Hank’s balanced salt solution (HBSS) containing 3% FBS before staining and fixation in 1% paraformaldehyde. Immune cell populations were identified by flow cytometry using an LSRII (BD Biosciences, San Jose, CA) and analyzed by FlowJo software (Tree Star, Ashland, OR). Lymph nodes or splenocytes were stained with anti-mouse CD3-FITC and anti-mouse CD44-APC purchased from eBioscience (San Diego, CA). Additionally, cells were stained for anti-mouse CD4-APC-Cy7, anti-mouse CD62L PeCy7, anti-mouse CD8-Pacific Blue, anti-mouse CD95-PE, anti-mouse CD19-PECy7, and anti-mouse GL-7 Pacific Blue (Biolegend, San Diego, CA).

Junkins R.D., Gallovic M.D., Johnson B.M., Collier M.A., Watkins-Schulz R., Cheng N., David C.N., McGee C.E., Sempowski G.D., Shterev I., McKinnon K., Bachelder E.M., Ainslie K.M, & Ting J.P. (2017). A robust microparticle platform for a STING-targeted adjuvant that enhances both humoral and cellular immunity during vaccination. Journal of controlled release : official journal of the Controlled Release Society, 270, 1-13.

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Phenotypic Characterization of Immune Cells

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After incubation with CPP-conjugated molecules, the cells were resuspended in 100 μl antibody mixture diluted in PBS and incubated at 4°C for 15 min. To discriminate each cell subset from the mixed cell population, different antibody combinations were used. For cell type analysis, anti-mouse CD4-PerCP-Cy5.5 (BioLegend, Clone:RM4-5), anti-mouse CD19-PE-Cy7 (BioLegend, Clone:6D5), anti-mouse NK1.1-APC (BioLegend, Clone:PK136), anti-mouse CD11b-PE-Cy7 (eBioscience, Clone:H57-597), anti-mouse CD11c-APC (eBioscience, Clone:N418), anti-mouse I-A/I-E-APC-Cy7 (BioLegend, Clone:M5/114.15.2), and anti-mouse F4/80-PerCP-Cy5.5 (BioLegend, Clone:BM8) were used. Also, anti-mouse CD16/32 (BioLegend, Clone:93) was used for blocking the Fc receptor. For activated T cell analysis, anti-mouse CD69-FITC (BioLegend, Clone:H1.2F3), anti-mouse CD4-APC (eBioscience, Clone:GK1.5), and anti-mouse CD8-PerCP-Cy5.5 (eBioscience, Clone:53–6.7) were used. For activated B cell analysis, anti-mouse CD86-PE-Cy5 (BioLegend, Clone:GL-1), anti-mouse IgG2a, kappa-isotype (BioLegend, Clone:RTK2758), and anti-mouse CD19-PE (BioLegend, Clone:6D5) were used.

Lim S., Lee J.A., Koo J.H., Kang T.G., Ha S.J, & Choi J.M. (2016). Cell Type Preference of a Novel Human Derived Cell-Permeable Peptide dNP2 and TAT in Murine Splenic Immune Cells. PLoS ONE, 11(5), e0155689.

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FITC and PE Labelled MD39 Tetramer Generation

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To generate FITC and PE labelled MD39 tetramers, biotinylated avi-tagged MD39 produced as previously mentioned were incubated with molar ratios of Streptavidin-FITC or Streptavidin-PE (BioLegend) for 30 minutes at room temperature 8, (link)51 (link) . Single cell suspensions from spleen or lymph nodes of the animals were first washed with PBS, then stained with live dead dye (fluorescent violet reactive, Thermo Fisher) diluted in PBS at room temperature for 10 minutes, followed by addition of mouse Fcblock (anti-mouse CD16/32, BioLegend) diluted 1:100 in 1% FBS/PBS and incubation at room temperature for additional 30 minutes. The cells were washed, and then incubated with 5ug/mL Streptavidin-MD39-FITC and Streptavidin-MD39-PE at room temperature for 45 minutes. Antibody mixtures (anti-mouse CD19-PE-Cy7, Anti-mouse IgD APC-Cy7 and Anti-mouse IgM BV711, BioLegend, each diluted 1:200 in 1% FBS/PBS) were then added to the cells without any intermediate washing steps, followed by incubation for another 45 minutes at room temperature. The cells were washed once with 1% FBS/PBS, and then resuspended at 10 million/mL concentration in 1% FBS/PBS for sorting with the FACS ARIA II instrument. CD19+ IgM-IgD-MD39-Tetramer-FITC+ MD39-Tetramer-PE+ cells were then sorted in bulk into 1.5mL
Eppendorf tube containing 1% FBS/PBS for down-stream cDNA prep with 10x genomics.

Xu Z., Walker S.N., Wise M.C., Chokkalingam N., Purwar M., Moore A., Tello‐Ruiz E., Wu Y., Majumdar S., Zaidi F.I., Reuschel E.L., Patel I., Obeirne A., Du J., Schultheis K., Gites L., Smith T.R., Mendoza J., Broderick K.E., Humeau L., Pallesen J., Weiner D.B., & Kulp D.W. (2021). Structural identification of neutralizing antibodies induced by nucleic acid encoded native-like trimer.

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