Pacific blue

Manufactured by BioLegend

Sourced in United States

Pacific Blue is a fluorescent dye used in flow cytometry applications. It has an excitation maximum at 410 nm and an emission maximum at 455 nm, allowing it to be detected using standard flow cytometry instrumentation.

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Lab products found in correlation

20 protocols using pacific blue

Immunophenotyping of Murine Immune Cells

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Cells from the peritoneum, bone marrow, and blood were incubated in PBS with 10% mouse serum (Sigma-Aldrich) for 20 minutes on ice to block nonspecific binding. Ly6G-phycoerythrin (PECy7), CD11b-PerCP Cy5.5, or Pacific Blue (BioLegend), 7/4-Alexa-647 (AbD Serotec) antimouse antibodies were added to the cell suspensions at concentrations recommend by the supplier and incubated on ice for 30 minutes in the dark. 11β-HSD1 sheep-derived antibody, generated in-house (23 (link)), was used in combination with donkey antisheep secondary antibody (Alexa Fluor 488; Invitrogen). Cells were treated with a fixation and permeabilization kit (Fix and Perm; Invitrogen) according to the manufacturer's instructions to allow for intracellular staining with the 11β-HSD1 antibody (Supplemental Figure 2). Blood and bone marrow cells were treated with BD lysis buffer (BD Biosciences) to eliminate red blood cells. Fluorescence was determined by FACScalibur using Cellquest (Becton Dickinson UK Ltd) or 5L LSR Fortessa using FACSDiva (Becton Dickinson UK Ltd) and analyzed using FlowJo software (Treestar).

Coutinho A.E., Kipari T.M., Zhang Z., Esteves C.L., Lucas C.D., Gilmour J.S., Webster S.P., Walker B.R., Hughes J., Savill J.S., Seckl J.R., Rossi A.G, & Chapman K.E. (2016). 11β-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice. Endocrinology, 157(7), 2928-2936.

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Monocyte Differentiation Markers

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LPS (Escherichia coli serotype O55:B5) was purchased from Sigma-Aldrich (Munich, Germany). Recombinant human M-CSF, IFNγ and IL-10 were obtained from Peprotech (Rocky Hill, NJ, USA). Recombinant human GM-CSF and IL-4 were from Novartis AG (Basel, Switzerland). The HLDA10 mAb panel, tested within the 10th Human Leukocyte Differentiation Antigen Workshop, in Wollongong, Australia, is detailed at http://www.hcdm.org. Alexa Fluor 647-conjugated monomeric streptavidin used at a final concentration of 2.5 μg ml⁻¹ was prepared in-house. Allophycocyanin-conjugated goat anti-mouse IgG+IgM Ab used at a final dilution of 1:2500 was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Control antibodies included: Pacific Blue-, FITC- or PE-labelled mouse IgG1 isotype control mAb, clone MOPC-21 (Biolegend, San Diego, CA, USA), Pacific Blue-conjugated CD14 mAb, clone MEM-18, unlabelled mouse IgG1 isotype control mAb, clone PPV06 (both from EXBIO, Prague, Czech Republic) and biotin-labelled mouse IgG1 isotype control mAb MCA928B (AbD Serotec, Oxford, UK).

Ohradanova-Repic A., Machacek C., Fischer M.B, & Stockinger H. (2016). Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel. Clinical & Translational Immunology, 5(1), e55-.

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Cytokine Profiling of PBMC Responses

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In the ICS assay, we stimulated PBMC of H1, H2, and H3 with CMV (pp65 and IE1) and EBV (EBNA1, LMP1, and BZLF1) and incubated for 6 h in 37°C. For staining, we used FITC (CD4, Cat. no. 317408; BioLegend, RRID:AB_571951), PE (CD107a, Cat. no. 328608; BioLegend, RRID: AB_1186040), PerCP (CD8a, Cat. no. 300922; BioLegend, RRID: AB_1575072), PE-Cy7 (IL2, Cat. no. 500326; BioLegend, RRID: AB_2125593), APC (TNFa, Cat. no. 502912; BioLegend, RRID:AB_315264), APC-Cy7 (IFNg, Cat. no. 502530; BioLegend, RRID: AB_10663412), Pacific Blue (CD3, Cat. no. 300330; BioLegend, RRID:AB_10551436), and LIVE/DEAD Aqua-Amcyan Antigen for staining cells. We employed BD FACSCanto (BD), and the sort logic was set by gating lymphocytes by forward scatter and side scatter and then gating on CD3⁺ CD4⁺ cells and CD3⁺ CD8⁺ cells. The dataset was analyzed by FlowJo software version.10.

Kashima Y., Kaneko K., Reteng P., Yoshitake N., Runtuwene L.R., Nagasawa S., Onishi M., Seki M., Suzuki A., Sugano S., Sakata-Yanagimoto M., Imai Y., Nakayama-Hosoya K., Kawana-Tachikawa A., Mizutani T, & Suzuki Y. (2022). Intensive single-cell analysis reveals immune-cell diversity among healthy individuals. Life Science Alliance, 5(7), e202201398.

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Quantifying Apoptosis through Caspase-3 and Annexin V Assays

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Caspase-3 activity was quantified as described previously [57 (link)]. Release of fluorescent 7-amino-4-methylcoumarin (AMC) after cleavage from Ac-DEVD-AMC was quantified over 4 h. Apoptosis was also quantified by flow cytometry with Annexin V and PI. For staining 1 × 10⁵ cells were incubated with 80 μg/ml PI and 9.6 μg/ml Annexin V conjugated to PacificBlue (No. 640917, Biolegend, San Diego, CA, USA) in Annexin V binding buffer (No. 422201, Biolegend) for 15 min at room temperature. Cells were analysed on a FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). 10000 cells per sample were analysed. Data analysis was performed using FCS Express 4 Flow software (De Novo Software, Los Angeles, CA, USA).

Hussmann M., Janke K., Kranz P., Neumann F., Mersch E., Baumann M., Goepelt K., Brockmeier U, & Metzen E. (2015). Depletion of the thiol oxidoreductase ERp57 in tumor cells inhibits proliferation and increases sensitivity to ionizing radiation and chemotherapeutics. Oncotarget, 6(36), 39247-39261.

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Flow Cytometry Analysis of Murine Immune Cells

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For flow cytometry, we purchased mAbs to murine CD4 (Pacific Blue and APC-H7 from Biolegend, PE-CF594 and APC-Cy7 from BD Pharmingen), Ki-67 (PerCP-Cy5.5, clone B56, BD Pharmingen), Foxp3 (PE and eFluor450, clone FJK-16s, eBioscience), CTLA4 (PE, BD Pharmingen), I-A/I-E (FITC, BD Pharmingen), CD11b (APC from eBioscience, Pe/Cy7 from Biolegend), Ly-6G (APC, Biolegend), Ly-6C (Pacific Blue, Biolegend), Gr-1 (APC, Biolegend), F4/80 (APC and PE, Biolegend), CD11c (APC/Cy7, Biolegend), CD39 (eFluor 660, eBioscience), CD103 (PE, Biolegend), CD127 (Brilliant Violet421, Biolegend), CCR9 (FITC, Biolegend), and Integrin α₄β₇ (APC, Biolegend). For the analysis of mesenteric lymph nodes, we used the Aqua LIVE/DEAD® Fixable Dead Cell Stain Kit to exclude false positive signals from dead and apoptotic cells, and then washed two times prior to antibody staining. We purchased EX-527 from Tocris Bioscience. Drugs were dissolved in dimethylsulfoxide (DMSO), and DMSO was used as a control.

Akimova T., Xiao H., Liu Y., Bhatti T.R., Jiao J., Eruslanov E., Singhal S., Wang L., Han R., Zacharia K., Hancock W.W, & Beier U.H. (2014). Targeting Sirtuin-1 Alleviates Experimental Autoimmune Colitis by Induction of Foxp3+ T-Regulatory Cells: Sirt1 targeting alleviates autoimmune colitis. Mucosal immunology, 7(5), 1209-1220.

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ALDH Activity Assay with MitoTracker

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5 × 10⁵ cells were re-suspended in 1 ml Assay buffer and 5 μl ALDEFLUOR reagent (Stem Cell Technologies) was added and mixed well. 0.5 ml of this cell suspension was transferred into a new tube with 5 μl of DEAB regent (Stem Cell Technologies). Tubes were incubated for 30 mins at 37°C before centrifugation and re-suspension in Assay Buffer (0.5 ml) for FACS analysis (BD, LSR Fortessa). When combined with MitoTracker Deep-Red (Molecular Probes, 0.125 μl/ml), both were added before 30 min incubation. Note the PDX model BB6RC50 was also co-stained with anti-mouse MHC Class I (H-2Kd) antibody conjugated with Pacific Blue (BioLegend, 116616), to exclude mouse cells in the ALDH analysis.

Farnie G., Sotgia F, & Lisanti M.P. (2015). High mitochondrial mass identifies a sub-population of stem-like cancer cells that are chemo-resistant. Oncotarget, 6(31), 30472-30486.

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Flow Cytometric Characterization of ucMSC

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Flow cytometric characterisation of control and treated ucMSC was performed by labelling with standard MSC markers, according to ISSCT criteria [41 (link)]: CD13 (PE-Cy7, BD Biosciences, San Jose, CA, USA), CD73 (PE, BD Pharmingen, San Diego, CA, USA), CD90 (APC, R&D Systems) and CD105 (FITC, R&D Systems), hematopoietic markers CD31 (PB, BD Biosciences), CD45 (APC-Cy, BD Biosciences) and the surface markers: HLA class I (Pacific Blue, Biolegend, San Diego, CA, USA), HLA class II (PERCP, BD Biosciences), CD362 (APC, R&D Systems), PD-L1 (PE, Biolegend), CD271 (PE-Cy7, BD Pharmingen), CD73 (PE, BD Pharmingen) and CD40 (PERCP, Biolegend).

de Witte S.F., Merino A.M., Franquesa M., Strini T., van Zoggel J.A., Korevaar S.S., Luk F., Gargesha M., O’Flynn L., Roy D., Elliman S.J., Newsome P.N., Baan C.C, & Hoogduijn M.J. (2017). Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease. Stem Cell Research & Therapy, 8, 140.

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Activation and Characterization of Human PBMCs

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Cryopreserved PBMCs from IRB consented blood donors were thawed at 37°C, washed twice with complete RPMI media [cRPMI; RPMI 1640 medium (Cellgro) supplemented with 5mM HEPES, 2mM Glutamax (Invitrogen), 50 μg/mL Penicillin (Invitrogen), 50 μg/mL Streptomycin (Invitrogen), 50 M 2-mercaptoethanol (Sigma-Aldrich), and 10% FBS (Atlanta Biologicals)], and plated at 2.5×10⁵ cells per well in cRPMI. Cells were activated with soluble anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL), and treated with IL-12 (1 ng/mL) and/or IL-18 (10 ng/mL) (R&D Systems) for 72h as described previously^{1 (link)}. GolgiStop (4 μL/6 mL culture; BD Biosciences) was added four hours prior to staining for flow cytometric analysis. Cells were first stained for the following surface markers: CD4–Pacific Blue (RPA-T4; eBioscience), TIGIT–APC or –PerCP-eFluor710 (MBSA43; eBioscience), CD226–PE (11A8; BD Biosciences), CD8–APC-Cy7 (SK1; BD Biosciences), and CD25–APC or –AlexaFluor (AF)-488 (BC96; BioLegend). For intracellular staining, cells were fixed, permeabilized, and stained for IFN-γ–PE-Cy7 (4S.B3; BioLegend), Helios–PE, –Pacific Blue, or –AF647 (22F6; BioLegend), and FOXP3–AF488 or –PE (206D; BioLegend) using the FOXP3 Fix/Perm staining kit (BioLegend) according to manufacturer’s protocol. Samples were collected on a BD LSRFortessa and analyzed using FlowJo software.

Dean J.W., Peters L.D., Fuhrman C.A., Seay H.R., Posgai A.L., Stimpson S.E., Brusko M.A., Perry D.J., Yeh W.I., Newby B.N., Haller M.J., Muir A.B., Atkinson M.A., Mathews C.E, & Brusko T.M. (2020). Innate Inflammation Drives NK Cell Activation to Impair Treg Activity. Journal of autoimmunity, 108, 102417.

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Flow Cytometry Immunophenotyping Protocol

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The following antibodies or streptavidin coupled to biotin, peridinin-chlorophyll-protein–Cy5.5, fluorescein isothiocyanate, AF488, allophycocyanin, AF647, Pacific Blue, Pacific Orange, allophycocyanin-Cy7, phycoerythrin, and phycoerythrin-Cy7 were purchased from BioLegend (San Diego, Calif), eBioscience (San Diego, Calif), BD Biosciences (San Jose, Calif), or Invitrogen (Carlsbad, Calif): CD19 (clone 6D5), B220 (RA3-6B2), CD3 (145-2C11), CD4 (RM4-5), CD11c (N418), CD8α (53-6.7), CD45.1 (A20), CD45.2 (104), CD44 (IM7), CXCR5 (2G8), MHC class II (M5/114.15.2), Siglec-F (E50-2440), CD11b (M1/70), CCR3 (TG14/CCR3), Gr-1 (RB6-8C5), CD80 (16-10A1), CD86 (GL1), CD40 (3/23), OX40 ligand (RM134L), CD43 (S7), CD5 (53-7.3), IgM (R6-60.2), and IgD (11-26c). Dead cells were excluded with 7-aminoactinomycin D (eBioscience). Flow cytometry was performed on either a FACSCanto II or BD Fortessa (BD Biosciences), and data were analyzed with FlowJo software (TreeStar, Ashland, Ore).

Wypych T.P., Marzi R., Wu G.F., Lanzavecchia A, & Sallusto F. (2017). Role of B cells in TH cell responses in a mouse model of asthma. The Journal of allergy and clinical immunology, 141(4), 1395-1410.

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Extracellular Vesicle Identification and Analysis

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Sample analysis solution for the MV was isotonic NaCl and KCl, with 10 mM HEPES (pH 7.3) and 3 mM Ca, plus 1% Annexin V conjugated to Pacific Blue (BioLegend 640918) to label surface phosphotidylserine (PS). The frozen MV samples were thawed and 45μl added to 225μl of analysis solution. A BD FacsCanto instrument was set so that 3000 nm polystyrene beads were at ~100,000 on forward and side scatter (FSC,SSC). Countable particles required both a FSC and SSC greater than 250. MV were identified as a distinct group of particles, much smaller than the reference beads and with high Annexin V staining.

Lao Z., Kelly C.J., Yang X.Y., Jenkins W.T., Toorens E., Ganguly T., Evans S.M, & Koch C.J. (2015). Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA. PLoS ONE, 10(7), e0133895.

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