Pacific blue anti mouse cd4

Manufactured by BioLegend

Sourced in United States

Pacific Blue anti-mouse CD4 is a fluorescence-conjugated antibody that binds to the CD4 protein expressed on the surface of mouse T helper cells. It can be used for the identification and enumeration of CD4+ T cells in flow cytometry applications.

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Lab products found in correlation

Fitc anti mouse cd3, by BioLegend (4 mentions) Apc anti mouse cd11b, by BioLegend (2 mentions) Fitc anti mouse ly 6g, by BioLegend (2 mentions) 7 amino actinomycin d viability staining solution, by BioLegend (2 mentions) Pacific blue anti mouse gr 1, by BioLegend (2 mentions) Apc cy7 anti mouse ly 6c, by BioLegend (2 mentions) Fitc anti mouse cd8α, by BioLegend (2 mentions) Facscanto 2 flow cytometer, by BD (2 mentions) Ionomycin, by Merck Group (2 mentions) Fitc anti mouse cd11c, by BioLegend (1 mentions) Pe anti mouse f4 80, by BioLegend (1 mentions) Trustain fcx anti mouse cd16 32 antibody, by BioLegend (1 mentions) Pe anti mouse nk 1, by BioLegend (1 mentions) Apc anti mouse cd3ε, by BioLegend (1 mentions) Streptavidin percp, by BD (1 mentions) Apc anti mouse tnf α, by BioLegend (1 mentions) Pe cy5 anti mouse cd3, by Thermo Fisher Scientific (1 mentions) Golgistop solution, by BD (1 mentions) Facscanto 2, by BD (1 mentions) Fitc anti mouse ifn γ, by BD (1 mentions)

9 protocols using pacific blue anti mouse cd4

Multiparametric Flow Cytometry Analysis of Immune Cell Subsets

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Cells were pelleted and washed in phosphate-buffered saline (PBS) supplemented with 2% FBS (2% FBS/PBS). The cell suspension was first blocked with TruStain fcX (anti-mouse CD16/32) antibody (BioLegend) for 5 min, and then stained with the following antibodies (Abs) for 15 min at 4°C: APC anti-mouse CD11b, Pacific Blue anti-mouse Gr-1, PE anti-mouse F4/80, FITC anti-mouse CD11c, APC-Cy7 anti-mouse Ly-6C, FITC anti-mouse Ly-6G, Pacific Blue anti-mouse CD4, and FITC anti-mouse CD8α (BioLegend). Next, the cells were washed and resuspended in 2% FBS/PBS. Shortly before performing measurements, a 7-amino actinomycin D viability staining solution (BioLegend) was added to each sample to stain dead cells. Flow cytometry analysis was performed on a BD FACSCanto II flow cytometer (BD Biosciences), and results were analyzed using the FlowJo software (version10.7.0, BD Biosciences).

Xie Z., Kawasaki T., Zhou H., Okuzaki D., Okada N, & Tachibana M. (2022). Targeting GGT1 Eliminates the Tumor-Promoting Effect and Enhanced Immunosuppressive Function of Myeloid-Derived Suppressor Cells Caused by G-CSF. Frontiers in Pharmacology, 13, 873792.

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Comprehensive Immunophenotyping of Murine Immune Cells

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Cells were pelleted, washed with 2% FBS/HBSS, blocked with TruStain fcX (anti-mouse CD16/32) antibodies (BioLegend, CA, USA) for 5 min, and then stained with the following antibodies for 15 min at 4°C: APC anti-mouse CD11b, Pacific Blue anti-mouse Gr-1, APC-Cy7 anti-mouse Ly-6C, FITC anti-mouse Ly-6G, APC anti-mouse CD3ε, Pacific Blue anti-mouse CD4, PE anti-mouse NK1.1, and FITC anti-mouse CD8α (BioLegend). The cells were then washed and resuspended in 2% FBS/HBSS. Shortly before performing measurements, a 7-amino actinomycin D viability staining solution (BioLegend) was added to each sample to stain dead cells. Flow cytometry analysis was performed using a BD FACSCanto II flow cytometer (BD Biosciences, NJ, USA). Data were analyzed using the FlowJo software (version 10.7.0, BD Biosciences). The gating strategy used for flow cytometry analysis was as follows: monocytes (7AAD⁻CD45⁺CD11b⁺Ly-6G⁻Ly-6C^hi), neutrophils (7AAD⁻CD45⁺CD11b⁺Ly-6G⁺Ly-6C^int), CD4⁺ T cells (7AAD⁻CD45⁺CD3ε⁺CD4⁺NK1.1⁻), CD8⁺ T cells (7AAD⁻CD45⁺CD3ε⁺CD8α⁺NK1.1⁻), and NK cells (7AAD⁻CD45⁺CD3ε⁻NK1.1⁺) (Figure S1A).

Xie Z., Zhou H., Obana M., Fujio Y., Okada N, & Tachibana M. (2023). Myeloid-derived suppressor cells exacerbate poly(I:C)-induced lung inflammation in mice with renal injury and older mice. Frontiers in Immunology, 14, 1243851.

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Cytokine Profiling of Splenocytes and Lung Cells

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Freshly isolated splenocytes or pulmonary immune cells were resuspended at a concentration of 1 × 10⁷cells/mL. Cells were stimulated with 10 μg/mL antigens (LppZ and Ag85A) or tuberculin PPD for 20 h or treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL Ionomycin (both from Sigma Aldrich) for 5 h. GolgiStop solution (BD Biosciences) was added to cell culture during the last 5 h of ex vivo stimulation. For surface staining, cells were incubated with PE-Cy5-anti-mouse CD3 (eBioscience), Pacific Blue-anti-mouse CD4 (Biolegend), APC-Cy7-anti-mouse CD8 antibodies, biotin-Annexin V and PerCP-streptavidin (BD Biosciences) for 30 min in the dark at 4°C. After washing with PBS containing 2% FBS, cells were fixed and permeated by the Cytofix/Cytoperm reagent from Fixation/Permeabilization kit (BD Biosciences). Cells were incubated with FITC-anti-mouse IFN-γ, PE-anti-mouse IL-2 (both from BD Biosciences), and APC-anti-mouse TNF-α (Biolegend) antibodies for 45 min at 4°C. Cells were washed, resuspended in PBS and acquired with a FACS CantoII flow cytometer (BD Bioscience) in 2 h. Data were analyzed by using FlowJo software 7.5 (Treestar Inc.).

Chen Y., Xiao J.N., Li Y., Xiao Y.J., Xiong Y.Q., Liu Y., Wang S.J., Ji P., Zhao G.P., Shen H., Lu S.H., Fan X.Y, & Wang Y. (2019). Mycobacterial Lipoprotein Z Triggers Efficient Innate and Adaptive Immunity for Protection Against Mycobacterium tuberculosis Infection. Frontiers in Immunology, 9, 3190.

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Multi-parameter Flow Cytometry Analysis of Immune Cells

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The spleen was dissociated into single cells by mashing the tissue through a
70-µm cell strainer. Red blood cells from spleens were lysed using 0.83% (w/v)
of ammonium chloride. For the detection of immune cell subtypes, the following
antibodies were used: Alexa Fluor 647 anti-mouse FOXP3 (Biolegend, 126407),
Alexa Fluor 700 anti-mouse CD45 (Biolegend, 103127), Brilliant Violet 510
anti-mouse CD8a (Biolegend, 100751), Brilliant Violet 650 anti-mouse CD19
(Biolegend, 115541), Brilliant Violet 785 anti-mouse Ly-6C (Biolegend, 128041),
FITC anti-mouse CD3 (Biolegend, 100203), Pacific Blue anti-mouse CD4 (Biolegend,
100427), PE anti-mouse IL-4 (Biolegend, 504103), PC/Cy7 anti-mouse IFNgamma
(Biolegend, 505825), PE/Dazzle 594 anti-mouse IL-17A (Biolegend, 506937), and
PerCP/Cy5.5 anti-mouse CD11b (Biolegend, 101227). Viable cells were gated using
the Zombie NIR Fixable Viability Kit (Biolegend, 423105). For cell surface
staining, cells were incubated with 10% rat serum for 15 min prior to staining
with fluorescently labeled antibodies for 15 min on ice. For intracellular
staining, cells were fixed, permeabilized, and stained using the Transcription
Factor Staining Buffer Set (Thermo Fisher Scientific) according to the
manufacturer’s protocol. Flow cytometry was carried out and analyzed on a BD
LSRFortessa (BD Biosciences).

Bogie J.F., Grajchen E., Wouters E., Broux B., Stinissen P., Van Wijmeersch B, & Hendriks J.J. (2020). CNS delivery of anti-CD52 antibodies modestly reduces disease severity in an animal model for multiple sclerosis. Therapeutic Advances in Chronic Disease, 11, 2040622320947378.

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Multiparametric Flow Cytometry of Immune Cells

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The mice were killed by CO₂ inhalation. To acquire single cells, lung was fragmented and digested with collagenase IV, and spleen was fragmented and filtered. Single‐cell suspensions of spleen and lung were incubated with FcR‐specific blocking mAb (eBioscience) and stained with FITC‐anti‐mouse CD3, Pacific blue‐anti‐mouse CD4, Brilliant Violet 605‐anti‐mouse CD8, APC‐anti‐mouse CD19, and Percp‐Cy5.5‐anti‐mouse CD335 (BioLegend, San Diego, CA, USA) and then examined by flow cytometry (LSRFortessa, BD Bioscience). The adhesion cells were excluded by FSC‐H/A and SSC‐H/A, and the lymphocytes were gated by FSC‐A and SSC‐A. The CD3⁺ cells were gated to determine the percentage of CD4⁺ and CD8⁺ cells, the CD3⁻ cells were gated to determine the percentage of CD19⁺ and CD335⁺ cells.

Zhu M., Niu B., Liu L., Yang H., Qin B., Peng X., Chen L., Liu Y., Wang C., Ren X., Xu C., Zhou X, & Li F. (2022). Development of a humanized HLA‐A30 transgenic mouse model. Animal Models and Experimental Medicine, 5(4), 350-361.

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Comprehensive T-cell Phenotyping by Flow Cytometry

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FITC anti-mouse CD3 (#100204, 1:200), Pacific Blue anti-mouse CD4 (#100531, 1:200), PerCP/Cyanine5.5 anti-mouse CD4 (#100434, 1:200), FITC anti-mouse CD8a (#100706, 1:200), PE/Cyanine7 anti-mouse CD8a (#100722, 1:200), APC anti-mouse IFN-γ (#505810, 1:200), PE anti-mouse TNF-α (#506306, 1:200), PE anti-mouse CD366 (Tim-3) (#119704, 1:200), and APC anti-mouse CD279 (PD-1) (#135210, 1:200) were ordered from Biolegend.
For intracellular cytokine staining, lymphocytes (1 × 10⁶) were stimulated with 50 ng/mL of PMA (phorbol 12-myristate 13-acetate) (#P8139, Sigma) and 1 μM of ionomycin (#13909, Sigma) in the presence of brefeldin A (#S7046, Selleck) (5 μg/mL) for 4 h. Then the stimulated cells were fixed and permeabilized with Fixation Buffer (#420801, Biolegend) for 20 min at 4°C, and stained with fluorochrome-conjugated antibody cocktails for 15 min at 4°C. Flow cytometry data were acquired on BD LSRFortessa X-20 and analyzed with FlowJo software.

Xie J., Wang S., Zhong Y., Gao M., Tian X., Zhang L., Pan D., Qin Q., Wu B., Lan K., Sun Z.J, & Zhang J. (2023). Oncolytic herpes simplex virus armed with a bacterial GBP1 degrader improves antitumor activity. Molecular Therapy Oncolytics, 29, 61-76.

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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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Nanoparticle-based Metformin Delivery System

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PLGA (lactide:glycolide: 75:25, M_w: 4,000-15,000) and organic solvents were purchased from Sigma (St. Louis, MO, USA). DPPC was purchased from Anatrace (Maumee, OH, USA). Metformin hydrochloride was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Agarose, ethidium bromide, and DNA gel loading dye (6x) for gel electrophoresis were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). Pharmaceutical grade chitosan oligosaccharide (M_w: 1.2 kD, 95% deacetylation) was purchased from Zhejiang Golden Schell Biochemical Co. Ltd (Zhejiang, China). The CCK-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Anti-POLR2A antibody, HRP-anti-rabbit Immunoglobulin G (IgG), HRP-anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Ki-67 antibody and anti-cleaved caspase-3 antibody were purchased from Cell Signalling (Danvers, MA, USA). Anti-GAPDH antibody was purchased from Abgent (San Diego, CA, USA). Alexa Fluor 488-labeled anti-mouse IgG antibody was purchased from Life Technologies (Waltham, MA, USA). Pacific Blue™ anti-mouse CD4 and APC/Cyanine7 anti-mouse CD8a antibodies were purchased from Biolegend (San Diego, CA, USA).

Xu J., Liu Y., Liu S., Ou W., White A., Stewart S., Tkaczuk K.H., Ellis L.M., Wan J., Lu X, & He X. (2022). Metformin Bicarbonate-Mediated Efficient RNAi for Precise Targeting of TP53 Deficiency in Colon and Rectal Cancers. Nano today, 43, 101406.

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Cytokine Profiling of Immune Cells

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The lymph node and spleen derived single cell suspensions were stimulated with 50 ng mL -1 PMA, 500 ng mL -1 ionomycin in the presence of 0.5 μL brefeldin A (to prevent the cells from secreting cytokines) (Biolegend, Switzerland) for 4 h at 37 °C, 5% CO 2 . The cell suspensions were washed with 0.5 mL of 1% fetal calf serum and centrifuged at 300 g for 5 min. Cells were stained according to manufacturer's instructions with antibodies for CD3 (PeCy7 anti-mouse CD3ε, Biolegend, CH) and CD4 (Pacific blue anti-mouse CD4, Biolegend, Switzerland). Following fixation with intracellular fixation buffer (Biolegend, CH) and permeabilization of the cells with Permeabilization buffer (Biolegend, CH), the lymph node cells were stained for IL-4 (anti-mouse IL-4 APC, eBiosciences, Switzerland), IL-10 (anti-mouse IL-10 PE, eBiosciences, CH), IL-17A (anti-mouse IL-17A Alexa Fluor 488, eBiosciences, Switzerland) and IFN-γ (anti-mouse IFN-γ PerCP-Cy5.5, eBiosciences, CH), according to manufacturer protocols. The cells were washed and re-suspended in MACS buffer (Mitenyi Coulter, Switzerland) and a Gallios flow cytometer (Beckman Coulter, Switzerland) was used to measure cellular fluorescence with data processing by Kaluza software (Beckman Coulter, Switzerland). The results were expressed as percentage of the total cell number.

Rochford E.T.J., Sabaté Brescó M., Zeiter S., Kluge K., Poulsson A., Ziegler M., Richards R.G., O'Mahony L, & Moriarty T.F. (2016). Monitoring immune responses in a mouse model of fracture fixation with and without Staphylococcus aureus osteomyelitis. Bone, 83.

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