Anti ccr7 pe cy7

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Anti-CCR7-PE-Cy7 is a fluorescently conjugated antibody that binds to the CCR7 (C-C chemokine receptor type 7) protein. CCR7 is a chemokine receptor that plays a role in lymphocyte trafficking and homing.

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CCR7-PE (26 citations) CCR7-PE-Cy7 (19 citations) Anti-CCR7 (13 citations) Anti-CCR7-PE (4 citations)

11 protocols using anti ccr7 pe cy7

Phenotypic Analysis of T Cell Subsets

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Cryopreserved PBMCs isolated from healthy controls (n=10) and day 1, day 7, day 28 and day 180 from patients (n=5-10/time point/disease category) were thawed, rested and stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) to exclude dead cells. To analyze the frequency of different Th cell subsets, PBMCs were stained with anti-CD3-AmCyan (clone SK7, BD), anti-CD4-APC (clone SK3, BD), anti-CD45RO- PerCP/Cyanine5.5 (Biolegend), anti-CCR7-PECy7 (Biolegend), anti-CXCR5-AF488 (RF8B2, BD Pharmingen), anti-CXCR3-PE (1C6/CXCR3, BD), anti-CCR6-BV421 (11A9, BD Horizon), anti-PD-1- PE-CF594 (EH12.1, BD) and anti-ICOS- BV605 (DX29, BD). Among the lymphocytes, 95% of cells were found to be alive as determined by live/dead staining. Different subsets of live T cells were analyzed following the gating strategy shown in Supplementary Figure 2.

Akhtar M., Basher S.R., Nizam N.N., Kamruzzaman M., Khaton F., Banna H.A., Kaisar M.H., Karmakar P.C., Hakim A., Akter A., Ahmed T., Tauheed I., Islam S., Ahmmed F., Mahamud S., Hasnat M.A., Sumon M.A., Rashed A., Ghosh S., Calderwood S.B., Harris J.B., Charles R.C., LaRocque R.C., Ryan E.T., Banu S., Shirin T., Chowdhury F., Bhuiyan T.R, & Qadri F. (2022). Longevity of memory B cells and antibodies, as well as the polarization of effector memory helper T cells, are associated with disease severity in patients with COVID-19 in Bangladesh. Frontiers in Immunology, 13, 1052374.

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Comprehensive Immune Cell Profiling

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The following mAbs were used: anti-CD20 BUV395, anti-CD10 APC, anti-CD4 BUV737, anti-CD4 APCCy7, anti-CD25 PECy7, anti-CD27 PECy7, anti-CD27 PE, anti-CD45RA BV605, anti-CXCR5 A647, anti-IgG APC, anti-IgG PE, anti-IgG BV605, anti-CD8 BUV395, anti-PD1 BV605, Streptavidin-PerCPCy5.5, anti-IgM PerCPCy5.5, anti-CD3 BV421, anti-IL-2 BV711, anti-IL-9 PerCPCy5.5, anti-IL-13 BV421, anti-IL-17F BV786, anti-IFN-γ BV605, anti-TNF-α BUV395, anti-CD19 BV711, anti-CD34 FITC, anti-CCR6 PE (all from Becton Dickinson); anti-CCR7 PECy7, anti-CD127 BV650, anti-IL-17A APCCy7, anti-CD20 Pacific Blue, anti-FoxP3 PE, anti-CXCR3 BV421 (BioLegend); anti-CD45RA PerCPCy5.5 (eBioscience); anti-CCR7 FITC (R&D Systems); anti-IgA-biotin (SouthernBiotech); anti-IL-4 PECy7, anti-IL-21 e660, anti-IL-22 PE (Thermo Fisher Scientific).

Nguyen T., Lau A., Bier J., Cooke K.C., Lenthall H., Ruiz-Diaz S., Avery D.T., Brigden H., Zahra D., Sewell W.A., Droney L., Okada S., Asano T., Abolhassani H., Chavoshzadeh Z., Abraham R.S., Rajapakse N., Klee E.W., Church J.A., Williams A., Wong M., Burkhart C., Uzel G., Croucher D.R., James D.E., Ma C.S., Brink R., Tangye S.G, & Deenick E.K. (2023). Human PIK3R1 mutations disrupt lymphocyte differentiation to cause activated PI3Kδ syndrome 2. The Journal of Experimental Medicine, 220(6), e20221020.

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Multiparameter Flow Cytometry Characterization

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After stimulation, cells were stained for surface and intracellular markers as
performed previously [23 (link), 24 (link)]. The following antibodies and dyes were
used; Aqua LIVE/DEAD (Invitrogen), anti-CD3 BUV396 (UCHT1; BD Biosciences),
anti-CD19 BV510 (SJ25C1; BioLegend), anti-CD14 BV510 (M5E2; BioLegend), anti-CD4
BUV805 (SK3; BD Biosciences), anti-CD8 BV786 (RPA-T8; BD Biosciences), anti-CCR7
PE/Cy7 (G043H7; BioLegend), anti-CD45RA PE/TR (MEM-56; Invitrogen), anti-KLRG1
eFlour710/PerCP (13F12F2; eBioscience), anti-IFN-γ AF700 (B27;
BioLegend), anti-IL-2 APC/Cy7 (MQ1-17H12; BioLegend), anti-TNF-α Pacific
Blue (MAb11; BioLegend), anti-Perforin-1 PE (B-D48; Cell Sciences),
anti-Granzyme B PE/Cy5.5 (GB11; Invitrogen), anti-T-bet BV605 (4B10; BioLegend),
and anti-Eomesodermin (EOMES) eFlour660 (WD1928; eBioscience). Flow cytometric
analysis was performed on a LSRFortessa (BD Biosciences) and analyzed by Flowjo
v10.6.1. Statistics analysis was done by SPICE V6 [25 (link)] and GraphPad Prism 8.

Jin W., Fang M., Sayin I., Smith C., Hunter J.L., Richardson B., Golden J.B., Haley C., Schmader K.E., Betts M.R., Tyring S.K., Cameron C.M., Cameron M.J, & Canaday D.H. (2023). Differential CD4+ T-Cell Cytokine and Cytotoxic Responses Between Reactivation and Latent Phases of Herpes Zoster Infection. Pathogens and Immunity, 7(2), 171-188.

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Cytotoxicity Assessment of Compounds in Tonsillar Tissues

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To assess the cytotoxicity of (1), (2), and (5) in human tonsillar tissues after 12 days of culture, cells isolated from untreated tissue blocks and from those treated with compounds were stained with combinations of the following fluorescence- labeled monoclonal antibodies: anti– CD3-QD605, anti–CD4-QD655, anti–CD8-QD705, anti–CD25-APC, anti–CD38-PE, anti–HLA-DR-APC-Cy7, anti–CXCR4-Brilliant violet 421, anti–CCR5-PR-Cy5 anti–CD45RA-FITC, and anti–CCR7-PE-Cy7 (Caltag Laboratories; Biolegend). Detection and enumeration of HIV-1– infected cells were performed with intracellular staining by means of anti–p24-PE (Beckman Coulter). Data were acquired and analyzed as described elsewhere (Grivel and Margolis, 2009 ). We quantified cell depletion using Trucount beads (Becton Dickinson) for volumetric control and normalized cell numbers by tissue-block weights.

Vanpouille C., Khandazhinskaya A., Karpenko I., Zicari S., Barreto-de-Souza V., Frolova S., Margolis L, & Kochetkov S. (2014). A NEW ANTIVIRAL: CHIMERIC 3TC-AZT PHOSPHONATE EFFICIENTLY INHIBITS HIV-1 IN HUMAN TISSUES EX VIVO. Antiviral research, 109, 125-131.

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Multiparametric Flow Cytometry Analysis

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Anti-CD44-PE, anti-CXCR4-PerCP/Cy5.5,
and anti-CCR7-PE/Cy7
antibodies were purchased from Biolegend. Cells (2.5 × 10⁵) were suspended in 100 μL of PBS and labeled by incubating
with antibodies (5 μL per test) at room temperature for 20 min
in the dark. Cells were washed twice, resuspended in PBS, and analyzed
on a CytoFLEX flow cytometry system. Data analysis was performed on
CytExpert software. Comparisons between treatment and control groups
were done with two-way ANOVA followed by Sidak’s multiple comparison
test. Differences in the percent changes in CD44, CXCR4, and CCR7
positive populations after treatment compared to their respective
control groups between the cell lines were analyzed by one-way ANOVA
followed by Tukey’s multiple comparison test. P values lower than 0.05 was considered as statistically significant
(**p < 0.01 and ****p < 0.0001).

Aru B., Günay A., Şenkuytu E., Yanıkkaya Demirel G., Gürek A.G, & Atilla D. (2020). A Translational Study of a Silicon Phthalocyanine Substituted with a Histone Deacetylase Inhibitor for Photodynamic Therapy. ACS Omega, 5(40), 25854-25867.

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Multiparametric Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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Specific antibodies against CD3-PE-Cy7 (Biolegend) or anti-CD3-FITC (Biolegend), anti-CD25-PE (BD Pharmingen™), anti-CD127-APC (Biolegend), anti-CD4-FITC (Biolegend), anti-CD8-APC (Biolegend), anti-CD45RA-FITC (Biolegend), anti-CCR7-PE-Cy7 (Biolegend), anti-CD4-PE (BD Biosciences), anti-PD-1-FITC (Biolegend), anti-CD4-PErCP-Cy5.5 (BD Biosciences), anti-Tim-3-PE (BD Pharmingen™), anti-FoxP3-PE (Biolegend), and anti-IFN-γ-APC (eBioscience) were used. 7AAD viability staining (Biolegend) was used to assess the viability of the cells. Tils were examined at day 0, 5, 10, 15, 20 and 25 by flow cytometry. Briefly, 1 × 10⁶ T cells was mixed with 5 μl of each antibody and was incubated on ice for 20 min in the dark. After incubation the samples were washed with FACS buffer (5% BSA in PBS, 0.09% sodium azide). The pellets were suspended in 300 μl of FACS buffer and acquired on a BD FACS Conto II flow cytometer, and then the data was analyzed with FlowJo software (TreeStar Inc). The data is displayed as background-corrected values with control sample or by fluorescence minus one (FMO).

Xu B., Yuan L., Chen G., Li T., Zhou J., Zhang C., Qin P., Muthana M.M., Wang S., Du X, & Gao Q. (2019). S-15 in combination of Akt inhibitor promotes the expansion of CD45RA−CCR7+ tumor infiltrating lymphocytes with high cytotoxic potential and downregulating PD-1+Tim-3+ cells as well as regulatory T cells. Cancer Cell International, 19, 322.

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Comprehensive Immune Profiling of Pancreatic Tumors

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Pancreatic tumour cell lines, MDDC and T-cells were assessed for expression of different markers using the following antibodies: Anti-PDL-1 APC, anti-Galectin 9 PE, anti-CD39 FITC, anti-CD47 Percp-Cy5.5, anti-HLA-class I Alexa Flour 700, anti-MIC A/B PE, anti-ULBP1 FITC, anti-ULBP 2,5 6 Percp, anti-ULBP 3 APC,anti-CD86 PE, anti-CCR7 PE-CY7, anti-CD40 APC, anti-HLA-class II PE-CY5 anti-IFN-γ PE-CY7, anti-CD3 Alexa Flour 488, anti-CD4 APC-CY7, anti-CD8-PE and anti-CD69 APC (Biolegend, CA).

Smith P.L., Yogaratnam Y., Samad M., Kasow S, & Dalgleish A.G. (2020). Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells. Clinical & Translational Oncology, 23(1), 110-121.

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Dendritic Cell Phenotypic Characterization

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The purity of generated DCs was assessed as CD14⁻CD11C⁺ by staining 1 × 10⁶ cells with anti-CD11C-APC and anti-CD14-FITC (BD Biosciences, San Diego, CA, USA).
Immature moDCs were resuspended to 1 × 10⁶ cells/ml and loaded with peptides±HB_100-108 (40 μg/ml for each peptide) or 40 μg/ml of free HB_100-108 at 37°C, 5% CO₂. After one hour, pulsed DCs were washed and cultured overnight. The following monoclonal antibodies (mAbs) were used to characterize the maturation and activation status of DCs: anti-HLA-ABC-PE, anti-HLA-DR-PE/Cy7, anti-CD80-PE, anti-CD86-APC, anti-CD83-APC, anti-CD40-Alexa Fluor 700, and anti-CCR7-PE/Cy7 (BioLegend, San Diego, CA, USA). Isotype-matched fluorescent antibodies were used as negative controls. Cells were incubated with antibodies at 4°C for 30 minutes. After washing, samples were detected by a FACSCalibur analyzer (BD Biosciences, San Jose, CA, USA). The data were analyzed by FlowJo software (TreeStar).

Chen C., Aldarouish M., Li Q., Liu X., Han F., Liu H, & Qian Q. (2020). Triggered Immune Response Induced by Antigenic Epitopes Covalently Linked with Immunoadjuvant-Pulsed Dendritic Cells as a Promising Cancer Vaccine. Journal of Immunology Research, 2020, 3965061.

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Multiparametric Flow Cytometry of PBMC Subsets

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Cryopreserved PBMCs and their subpopulations were analysed with a 28-color flow cytometry panel, as previously described [34 (link)]. The following mAbs were used: anti-CD20 BUV805, anti-CD10 APC, anti-Vαβ TCR BUV737, anti-CD4 APCCy7, anti-CD25 PECy7, anti-CD27 PECy7, anti-CD27 PE, anti-CD45RA PerCpCy5, anti- CXCR5 BUV615, anti-IgG APC, anti IgG BB660, anti-IgD BV480, anti-IgG BV605, anti-IgA1/A2 PECy5, anti-CD8 BUV496, anti-CD21 BUV563, anti-PD1 BV605, anti-IgM PerCPCy5.5, anti-IgM APC R700, anti-CD3 BV421, anti-IL-2 BV711, anti-IL-9 PerCPCy5.5, anti-IL-13 BV421, anti-IL-17F BV786, anti-IFN-γ BV605, anti-TNF-α BUV395, anti-CD19 BV711, anti-CD34 FITC, anti-CCR6 PE, anti-CD45RA BUV395, anti-CXCR5 BV615 (all from Becton Dickinson); anti-CD20 Pacific Blue, anti-CCR7 PECy7, anti-CD127 BV650, anti-IL-17A APCCy7, anti-CD20 BUV805, anti-CXCR3 BV421, anti-CD3 BV570 (BioLegend); (OptiBuild); anti-CCR7 FITC (R&D Systems); anti-IL-4 PECy7, anti-IL-21 e660, anti-IL-22 PE (Thermo Fisher Scientific).

Mina E.D., Jackson K.J., Crawford A.J., Faulks M.L., Pathmanandavel K., Acquarola N., O’Sullivan M., Kerre T., Naesens L., Claes K., Goodnow C.C., Haerynck F., Kracker S., Meyts I., D’Orsogna L.J., Ma C.S, & Tangye S.G. (2024). A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome. Journal of Clinical Immunology, 44(3), 66.

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Multiparameter Flow Cytometry Immunophenotyping

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The following antibodies were used for the flow cytometric analysis: anti-CD57-FITC, anti-CD28-PE, anti-CD4-PerCP, anti-CD8α-PerCP, anti-CCR7-PE-Cy7, anti-CD27-APC, anti-CD3-APC-Cy7, anti-CD45RA-APC-Cy7, anti-CD3-Pacific Blue, and anti-CD3-Pacific Blue (BioLegend, U.S.). A LIVEDEAD™ Fixable Aqua Dead Cell Stain kit (Thermo Fisher Scientific, U.S.) was used to monitor the cell viability. All flow data were acquired using FACS Canto II (Becton Dickinson), and the analysis was performed using FlowJo software ver.10.8.0 (Tree Star, U.S.).

Kashima Y., Mizutani T., Nakayama-Hosoya K., Moriyama S., Matsumura T., Yoshimura Y., Sasaki H., Horiuchi H., Miyata N., Miyazaki K., Tachikawa N., Takahashi Y., Suzuki T., Sugano S., Matano T., Kawana-Tachikawa A, & Suzuki Y. (2023). Multimodal single-cell analyses of peripheral blood mononuclear cells of COVID-19 patients in Japan. Scientific Reports, 13, 1935.

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