Ccr7 pe cy7 clone 3d12

Manufactured by BD

CCR7-PE-Cy7 (clone 3D12) is a fluorescently labeled antibody that binds to the chemokine receptor CCR7. It is a useful tool for the identification and analysis of CCR7-expressing cells in flow cytometry applications.

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6 protocols using ccr7 pe cy7 clone 3d12

Longitudinal Immune Profiling of GI and Lymphoid Tissues

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At the indicated longitudinal time points, lymph nodes were collected from peripheral sites (axillary, inguinal) and flash frozen. Total genomic DNA and RNA were prepared from these tissue samples as described above. Gastrointestinal biopsies from upper GI (duodenum/jejunum) and lower GI (colon) were collected as described [24 (link)], and dissociated in RPMI media containing 0.5 mg/mL collagenase and 1 U/mL DNase I. Viability of single cell suspensions was measured using a Guava Cytometer (Merck Millipore, Billerica, MA), and a small aliquot was stained with antibodies including CD3-Ax700 clone SP34-2, CD4-PerCP-Cy5.5 clone L200, CD8-APC-Cy7 clone SK1, CD28-PE-Cy5 clone CD28.2, CD45RA-FITC clone 5H9, CD95-APC clone DX2, CCR7-PE-Cy7 clone 3D12, and CCR5-PE clone 3A9, all from Becton Dickinson (Franklin Lakes, NJ). Total genomic DNA was extracted from the remainder of the sample for SHIV RNA and/or DNA analyses. When nucleic acid yields from these samples were insufficient, flash-frozen GI biopsy pinches collected at the same points were utilized.

Peterson C.W., Wang J., Deleage C., Reddy S., Kaur J., Polacino P., Reik A., Huang M.L., Jerome K.R., Hu S.L., Holmes M.C., Estes J.D, & Kiem H.P. (2018). Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy. PLoS Pathogens, 14(4), e1006956.

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Immunosenescence Assessment by Flow Cytometry

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Flow cytometry was performed on blood collected at baseline in ethylenediaminetetraacetic acid tubes (Sarstedt, Nümbrecht, Germany) on a FACSCanto II flow cytometer (Becton Dickinson, San Jose, California) using a single 8-color reagent panel (20 000–150 000 events per sample). Methods for sample preparation and instrument set-up were in accordance with the recommendations of the EuroFlow consortium [19 (link)]. Monoclonal antibodies used were: CD3 (APC, clone SK7), CD4 (HV450, clone RPA-T4), CD8 (APC-H7, clone SK1), CD28 (FITC, clone CD28.2), CD45 (HV500, clone HI30), CD45RA (PerCP-Cy5.5, clone HI100), CD57 (PE, clone NK-1), and CCR7 (PE-Cy7, clone 3D12) (all Becton Dickinson). Data were analyzed using FACSDiva software (Becton Dickinson). The following subsets were used to assess immune senescence: CD4/CD8 ratio, proportion of memory CD4 and CD8 T cells (sum of the frequencies of CD45RA⁻CCR7⁺, CD45RA^–CCR7^–, and CD45RA⁺CCR7^– subpopulations), frequency of CD28^null T cells, and frequency of CD57⁺CD8 T cells. A detailed gating strategy for the various cell subset is shown in Supplementary Figure 4.

Van Praet J.T., De Groote M., De Bacquer D., Verhalleman E., Welvaert Z., Emmerechts J., Reynders M, & De Vriese A.S. (2022). Immune Senescence Markers Predict the Cellular Immune Response to BNT162b2 Vaccination in Hemodialysis Patients. Open Forum Infectious Diseases, 9(11), ofac585.

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Quantifying CD4+ T Cell Subsets

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CD4+ T cell subsets were sorted on a BD FACSAria II cell sorter. The following antibodies were used: CD3-FITC (clone HIT3a, BD #555339), CD4-APC (clone SK3, BD #340443), CD45RA-APCH7 (clone HI100, BD #560674), CCR7-PE-Cy7 (clone 3D12, BD #557648), CD27-PE (clone M-T271, BD #555441) and LIVE/DEAD fixable Aqua (Invitrogen #L34957). Frequencies of memory CD4+ T cell subsets were determined as previously described after the exclusion of dead cells (LIVE/DEAD) (55 (link)). Four CD4+ T cell subsets were defined based on the expression of CD45RA, CCR7 and CD27: naïve (T_N: CD45RA+, CCR7+, CD27+), central memory (T_CM: CD45RA−, CCR7+, CD27+), transitional memory (T_TM: CD45RA−, CCR7−, CD27+) and effector memory (T_EM: CD45RA−, CCR7−, CD27−). Integrated HIV DNA was measured in the sorted populations. All subsets for which insufficient number of cells were analyzed (<40,000 sorted cells as measured in the PCR assay) were excluded from the analysis.

Leyre L., Kroon E., Vandergeeten C., Sacdalan C., Colby D.J., Buranapraditkun S., Schuetz A., Chomchey N., de Souza M., Bakeman W., Fromentin R., Pinyakorn S., Akapirat S., Trichavaroj R., Chottanapund S., Manasnayakorn S., Rerknimitr R., Wattanaboonyoungcharoen P., Kim J.H., Tovanabutra S., Schacker T.W., O’Connell R., Valcour V.G., Phanuphak P., Robb M.L., Michael N., Trautmann L., Phanuphak N., Ananworanich J, & Chomont N. (2020). Abundant HIV-infected cells in blood and tissues are rapidly cleared upon ART initiation during acute HIV infection. Science translational medicine, 12(533), eaav3491.

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Multicolor Flow Cytometry of PBMCs

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Multicolor flow cytometric analysis was performed on whole blood or frozen PBMCs using predetermined optimal concentrations of the following fluorescently conjugated mAbs: CD3-PacBlue or -APC-Cy7 (clone SP34-2), CD95-PE-Cy5 (clone DX2), Ki-67-AF700 (clone B56), HLA-DR-PerCP-Cy5.5 (clone G46-6), CCR7-PE-Cy7 (clone 3D12), CCR5-PE or -APC (clone 3A9), CD45RA-FITC (clone L48), Biotin-CD122 (clone Mik-β3) from BD Biosciences; CD8-BV711 (clone RPA-T8), CD4-APC-Cy7 or -BV650 (clone OKT4), Streptavidin-PE from Biolegend, and CD28-ECD (clone CD28-2) from Beckman-Coulter. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on an LSRII flow cytometer driven by the FACSDiva software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo Version 10.0.4 software (TreeStar).

Mavigner M., Watkins B., Lawson B., Lee S.T., Chahroudi A., Kean L, & Silvestri G. (2014). Persistence of Virus Reservoirs in ART-Treated SHIV-Infected Rhesus Macaques after Autologous Hematopoietic Stem Cell Transplant. PLoS Pathogens, 10(9), e1004406.

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Multiparameter Flow Cytometry for T Cell Analysis

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For cell sorting, peripheral CD4⁺ T cells were first enriched by negative selection with the use of magnetic beads and column purification (nonhuman primate CD4⁺ T cell isolation kit; Miltenyi). Enriched CD4⁺ T cells were then stained with viability dye (Live/Dead Aqua) and previously determined volumes of the following fluorescently conjugated MAbs: CD3-AF700 (clone SP34-2), CD8-APC-Cy7 (clone SK1), CD95-PE-Cy5 (clone DX2), CD62L-PE (clone SK11), and CCR7-PE-Cy7 (clone 3D12) from BD Biosciences; CD4-BV650 from BioLegend. Sorted live CD3⁺CD8-CD4⁺ populations were defined as follows: naive cells, CD62L⁺ CCR7⁺ CD95-; and memory, CD95⁺. Sorting was performed on a FACSAria LSR II (BD Biosciences) equipped with FACSDiva software.

Bricker K.M., Obregon-Perko V., Williams B., Oliver D., Uddin F., Neja M., Hopkins L., Dashti A., Jean S., Wood J.S., Ehnert S., Liang S., Vanderford T., Tharp G.K., Bosinger S.E., Schauer A.P., Mavigner M., Cottrell M.L., Margolis D., Dunham R.M, & Chahroudi A. (2022). Altered Response Pattern following AZD5582 Treatment of SIV-Infected, ART-Suppressed Rhesus Macaque Infants. Journal of Virology, 96(7), e01699-21.

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Multiparameter Flow Cytometry of Immune Cells

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Cells were stained for 30 minutes at 4 0 C in PBS containing 0.01% NaN3 and 0.5% BSA (Sigma Aldrich) with directly labelled antibodies against: CXCR5 alexa fluor 488 (clone RF8B2), CCR7 PE-Cy7 (clone 3D12), CD4 APC-H7 (clone SK3), CD8 V450 (RPA-T8), CD3 V500 (UCHT1) (all from BD Biosciences, San Jose, CA), PD-1 PE (J105), CD45 RA (L307.4) efluor450 (all from eBioscience Inc., San Diego, CA). For intracellular cytokine staining we used IL-10 Pe-Cy7 (clone JES3-9D7) (Biolegend, San Diego, CA) and IL-21 alexa fluor 647 (clone 3A3-N2.1) (BD Biosciences). Cells were analyzed on a FACS Canto II (BD Biosciences) and data were analyzed using FlowJo software (FlowJo, Ashland, OR). Data were plotted as frequency of positive cells.

Anang D.C., Ramwadhdoebé T.H., Hähnlein J., Kuijk B.v., Smits N., Lienden K.P.v., Maas M., Gerlag D.M., Tak P.P., Vries N.d., & Baarsen L.G.M.v. (2021). Increased frequency of CD4⁺ and CD8⁺ follicular helper T cells in human lymph node biopsies during the earliest stages of rheumatoid arthritis.

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