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Cd14 v450

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The CD14-V450 is a fluorochrome-conjugated antibody used for the identification and enumeration of CD14-positive cells in flow cytometry analysis. It binds to the CD14 surface antigen, which is expressed on monocytes, macrophages, and other myeloid cells. The V450 fluorochrome allows for detection in the violet laser excitation range.

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Lab products found in correlation

Foxp3 apc, by Thermo Fisher Scientific (3 mentions) Cd8 apc cy7, by BD (3 mentions) Cd11b pe, by Beckman Coulter (3 mentions) Hla dr pc7, by Beckman Coulter (3 mentions) Cd15 fitc, by Beckman Coulter (3 mentions) Cd33 apc, by Beckman Coulter (3 mentions) Hla dr percp cy5, by BD (3 mentions) Viability dye, by Thermo Fisher Scientific (2 mentions) Hla dr apc cy7, by BioLegend (2 mentions) Cd4 bv605, by BD (2 mentions) Ki67 apc, by BD (2 mentions) Hla dr pe cy5, by BD (2 mentions) Ccr5 pe, by BD (2 mentions) Cd69 alexa fluor 700, by BioLegend (2 mentions) Cd38 fitc, by STEMCELL (2 mentions) Cd8 bv785, by BD (2 mentions) Cd11b pe cy5, by BioLegend (2 mentions) Cd14 bv711, by BioLegend (2 mentions) Cd8 bv785, by BioLegend (2 mentions) Cd25 bv421, by BioLegend (2 mentions)

15 protocols using cd14 v450

1

Rectal Immune Cell Characterization

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Single-cell suspensions from rectal pinches were prepared as previously described30 (link). Rectal intraepithelial lymphocytes (IEL) and lamina propria(LP) were collected and subjected to flow cytometry analysis. The single-cell suspensions were first incubated with Fc Receptor blocking reagent (Miltenyi Biotec), followed by staining with viability dye (Invitrogen). The antibody mixtures were then incubated as previously described27 (link). For immune activation, the following antibodies were used: CD45-PerCP, CD3-PE-Cy7, CD4-BV605, CD8-APC-Cy7, CD14-V450, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (BD Pharmingen); CD69-Alexa Fluor 700 (Biolegend); and CD38-FITC (STEMCELL Technologies). For detection of Treg and MDSCs, the following antibody mixture were used: CD45-PerCP/Cy5.5, CD3-PE-Cy7, CD4-BV605, CD8-BV785, lin 1-FITC (BD Pharmingen), FOXP3-APC (eBioscience), HLA-DR-APC-Cy7, CD11b-PE-Cy5, CD14-BV711, CD8-BV785, CD25-BV421, CD15-Alexa700 (Biolegend), CD33-PE (Milteny). An LSRII flow cytometer was used for data acquisition. FlowJo software (Tree Star Inc.) was used for data analyses.
Sui Y., Dzutsev A., Venzon D., Frey B., Thovarai V., Trinchieri G, & Berzofsky J.A. (2018). Influence of gut microbiome on mucosal immune activation and SHIV viral transmission in naive macaques. Mucosal immunology, 11(4), 1219-1229.
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2

Quantifying Myeloid-Derived Suppressor Cells

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PBMC from each patient at the pre-treatment and post-Cycle 1 time points were analyzed for myeloid derived suppressor cells (MDSC) as previously described [25 (link)]. Specific antibodies included CD15-FITC (Beckman Coulter), CD33-APC (Beckman Coulter), HLA-DR-PC7 (Beckman Coulter), CD11b-PE (Beckman Coulter), and CD14-V450 (BD Biosciences). Single color staining was performed for compensation. All samples were run on a BD LSR II flow cytometer and were subsequently analyzed with FlowJo software (TreeStar). MDSC were defined as cells positive for CD33 and CD11b and lacking HLA-DR with subsets expressing CD15 or CD14 representing granulocytic and monocytic MDSC, respectively, as discussed in figure legends.
Duggan M.C., Jochems C., Donahue R.N., Richards J., Karpa V., Foust E., Paul B., Brooks T., Tridandapani S., Olencki T., Pan X., Lesinski G.B., Schlom J, & Carson WE I.I.I. (2016). A phase I study of recombinant (r) Vaccinia-CEA(6D)-TRICOM and rFowlpox-CEA(6D)-TRICOM vaccines with GM-CSF and IFN-α-2b in patients with CEA expressing carcinomas. Cancer immunology, immunotherapy : CII, 65(11), 1353-1364.
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3

Multiparametric Phenotyping of Cervical Immune Cells

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Human cervical tissue was obtained from two sets of five healthy women (age range 42–47 years old) undergoing hysterectomy for benign indication at HUGTP. After confirmation of healthy tissue status by the Pathological Anatomy Service, a piece from ecto and endocervix separated by anatomical localization was delivered to the laboratory in refrigerated RPMI 1640 medium (Cellgro, Manassas, VA) containing 10% FBS (Lonza, Basel, Switzerland), 500U/mL penicillin, 500μg/mL streptomycin, 5μg/mL fungizone and 1μg/mL gentamycin (Life Technologies). Tissue was processed within the next 12 h after surgery, and 8-mm3 block-dissection was performed as described [26 (link)]. Tissue digestion of 5–9 pieces of ecto or endocervix with collagenase IV (Invitrogen) was immediately executed as described [26 (link)]. Tissue blocks were then dissociated manually with a disposable pellet pestle and filtered through a 70μm-cell strainer (BD Biosciences). After centrifugation, pellet was suspended in staining buffer (1% mouse serum, 1% goat serum in PBS) and stained with different combinations of CD3-eFluor 605 (eBiosciences), CD14-V450, CD11c-PE-Cy7, CD8-V500, HLA-DR-PerCP-Cy5.5, CD69-Horizon PE-CF594 (BD Biosciences), NK1.1-PE, γδTCR-FITC, Vα7.2-APC-H7 (Miltenyi Biotec), CD103-FITC and Vα24-APC (BioLegend). Data were acquired and analyzed as described for blood.
Qualai J., Li L.X., Cantero J., Tarrats A., Fernández M.A., Sumoy L., Rodolosse A., McSorley S.J, & Genescà M. (2016). Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential. PLoS ONE, 11(4), e0154253.
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4

Rectal Immune Cell Characterization

Cited 2 times
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Single-cell suspensions from rectal pinches were prepared as previously described30 (link). Rectal intraepithelial lymphocytes (IEL) and lamina propria(LP) were collected and subjected to flow cytometry analysis. The single-cell suspensions were first incubated with Fc Receptor blocking reagent (Miltenyi Biotec), followed by staining with viability dye (Invitrogen). The antibody mixtures were then incubated as previously described27 (link). For immune activation, the following antibodies were used: CD45-PerCP, CD3-PE-Cy7, CD4-BV605, CD8-APC-Cy7, CD14-V450, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (BD Pharmingen); CD69-Alexa Fluor 700 (Biolegend); and CD38-FITC (STEMCELL Technologies). For detection of Treg and MDSCs, the following antibody mixture were used: CD45-PerCP/Cy5.5, CD3-PE-Cy7, CD4-BV605, CD8-BV785, lin 1-FITC (BD Pharmingen), FOXP3-APC (eBioscience), HLA-DR-APC-Cy7, CD11b-PE-Cy5, CD14-BV711, CD8-BV785, CD25-BV421, CD15-Alexa700 (Biolegend), CD33-PE (Milteny). An LSRII flow cytometer was used for data acquisition. FlowJo software (Tree Star Inc.) was used for data analyses.
Sui Y., Dzutsev A., Venzon D., Frey B., Thovarai V., Trinchieri G, & Berzofsky J.A. (2018). Influence of gut microbiome on mucosal immune activation and SHIV viral transmission in naive macaques. Mucosal immunology, 11(4), 1219-1229.
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5

Multiparametric Flow Cytometry Analysis

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Multi-color flow cytometry analysis was performed on PBMCs from all time points by staining for 30 minutes at 4°C with CD3-V450, CD8-FITC or APC, ICOS-PE, HLA-DR-PerCP-Cy5.5, CD25-PE-Cy7, CD45RA-PerCP-Cy5.5, CD62L-FITC, CD127-V450, PD-1-PE, Tim-3-AF700, CD4-APC-Cy7 (BD Biosciences, San Jose, CA), CCR7-PE-Cy7 (R&D Systems, Minneapolis, MN), CTLA-4-FITC (LSBio, Seattle, WA) and FoxP3-APC (eBioscience, San Diego, CA) for T cells. For natural killer (NK) cells, CD3-V450, CD16-APC-Cy7, CD56-PE-Cy7 and Tim-3-AF700 (BD) were used. For myeloid-derived suppressor cells (MDSCs), CD33-PE, CD11b-APC-Cy7, HLA-DR-PerCP-Cy5.5, CD14-V450 and CD15-APC (BD) were used. 1×105 cells were acquired on an LSRII (BD), and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). The appropriate isotype controls were used, and dead cells were excluded from the analysis.
Jochems C., Tucker J.A., Tsang K.Y., Madan R.A., Dahut W.L., Liewehr D.J., Steinberg S.M., Gulley J.L, & Schlom J. (2014). A Combination Trial of Vaccine Plus Ipilimumab in Metastatic Castration-Resistant Prostate Cancer Patients: Immune Correlates. Cancer immunology, immunotherapy : CII, 63(4), 407-418.
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6

Flow Cytometry for Immune Cell Analysis

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For flow cytometry, the following antibodies were purchased from BD Biosciences: CD25-APC (PC61), CD3-FITC (17A2), CD3-APC-Cy7, CD4-PE (L3T4), CD8a-FITC (LY-2, 53-6.7), CD8a-APC (53-6.7), B220-PE (CD45RA-14.8), Annexin V-APC, CD45-V500, CD14-V450, IgG2a-PE rat isotype (R35–95), IgG2a-APC rat isotype), IgG2a-FITC, IgG2a-V500, IgG2a-V450 rat isotype. CD44-PE (IM7) was purchased from Biolegend, San Diego, CA, USA. Thymocytes, splenocytes and whole blood cells (1 × 106 cells) were incubated with fluorochrome-conjugated antibodies (0.5 μg) for 30 minutes on ice, avoiding light. After two PBS washes, cells were analysed by flow cytometry using BD FACSAria (BD Biosciences) flow cytometer and BD FACSDiva software (BD Biosciences), as previously described76 (link). Splenic B-cells (B220+), splenic T-cells (CD3+), splenic CD4+ and CD8+ cells were sorted by antigenic criteria using BD FACSAria flow cytometer/cell sorter, with a purity grade of 99%.
Fiume G., Scialdone A., Albano F., Rossi A., Maria Tuccillo F., Rea D., Palmieri C., Caiazzo E., Cicala C., Bellevicine C., Falcone C., Vecchio E., Pisano A., Ceglia S., Mimmi S., Iaccino E., Laurentiis A.D., Pontoriero M., Agosti V., Troncone G., Mignogna C., Palma G., Arra C., Mallardo M., Maria Buonaguro F., Scala G, & Quinto I. (2015). Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice. Scientific Reports, 5, 13864.
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7

MDSC Phenotyping in PBMC

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PBMC were analyzed for the presence of MDSC as previously described [21 (link)]. MDSC were defined as cells positive for CD33, CD11b and lacking HLA-DR with subsets expressing CD15 or CD14 representing granulocytic and monocytic MDSC, respectively (Fig. 1). Notably, the current method for phenotyping M-MDSC (CD33+/HLADR-/CD14+/CD11b+) compares favorably to methods employed by other investigators (e.g., CD14+/HLADRlow/−) with respect to the percentages of M-MDSC obtained [22 (link)]. Specific antibodies included CD15-FITC, CD33-APC, HLA-DR-PC7, CD11b-PE (all Beckman Coulter), and CD14-V450 (BD Biosciences). All samples were run on a BD LSR-II flow cytometer and data was analyzed with FlowJo software (Tree Star, Inc.).
Wesolowski R., Duggan M.C., Stiff A., Markowitz J., Trikha P., Levine K.M., Schoenfield L., Abdel-Rasoul M., Layman R., Ramaswamy B., Macrae E.R., Lustberg M.B., Reinbolt R.E., Mrozek E., Byrd J.C., Caligiuri M.A., Mace T.A, & Carson WE I.I.I. (2017). Circulating myeloid derived suppressor cells increase in patients undergoing neo-adjuvant chemotherapy for breast cancer. Cancer immunology, immunotherapy : CII, 66(11), 1437-1447.
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8

Multi-Dimensional Immune Profiling

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Multi-color flow cytometry analysis was performed on PBMCs of 20 patients from all time points by staining for 30 minutes at 4°C with CD3-V450, CD8-FITC or APC, HLA-DR-PerCPCy5.5, CD25-PECy7, CD45RA-PerCP-Cy5.5, CD62L-FITC, CD127-V450, CCR7-PE-Cy7, PD-1-PE, CD4-APC-Cy7, CTLA-4-FITC, and FOXP3-APC (BD Biosciences, San Jose, CA). For NK cells CD3-V450, CD16-APC-Cy7, CD56-PE-Cy7 and Tim-3-AF700 were used. For MDSC CD33-PE, CD11b-APC-Cy7, HLA-DR-PerCP-Cy5.5, CD14-V450 and CD15-APC (BD Biosciences) were used. 1×105 cells were acquired on an LSR-II (BD Biosciences), and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). The appropriate isotype controls were used, and dead cells were excluded from the analysis.
Farsaci B., Jochems C., Grenga I., Donahue R.N., Tucker J.A., Pinto P.A., Merino M.J., Heery C.R., Madan R.A., Gulley J.L, & Schlom J. (2014). Identification by digital immunohistochemistry of intratumoral changes of immune infiltrates after vaccine in the absence of modifications of PBMC immune cell subsets. International journal of cancer, 135(4), 862-870.
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9

CFSE-labeled PBMC Activation Assay with CM

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PBMC were labeled with carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes/ Thermo Fisher Scientific) as described elsewhere33 (link). 3 × 105 labeled PBMC were seeded in 400 μL complete RPMI (RPMI with 10% human AB-serum, 1% P/S and 1% glutamine) in 48-well plates. To induce a basal level of activation, PBMC were stimulated with a low dose (12.5 ng/mL) of αCD3 antibody (OKT3; Janssen-Cilag, Neuss, Germany). CFC and IFC tissue CM was made by incubating 8 mm tissue punches for 6 days in complete RPMI. Four hundred µL of CFC- or IFC-CM was added to the αCD3 stimulated PBMC culture. PBMC cultures without αCD3 stimulation served as control. After 4 days culture, PBMC were harvested with trypsin (Gibco/Thermo Fisher Scientific) and stained with human specific antibodies: CD8-PE (1:100) (Miltenyi Biotec), CD4-APC (1:100), CD14-V450 (1:1000) (BD Biosciences) and a viability marker in the V510 channel (1:400) (LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit; Invitrogen/Thermo Fisher Scientific). FACS staining and measurement was performed as described above.
Schneider M., Stamm C., Brockbank K.G., Stock U.A, & Seifert M. (2017). The choice of cryopreservation method affects immune compatibility of human cardiovascular matrices. Scientific Reports, 7, 17027.
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10

Characterization of Dendritic Cell Subsets

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Cells from each culture condition were incubated with monoclonal antibodies for 20 min at room temperature in the dark. The following monoclonal antibodies were used: CD11c PE-Cy7, CD14 V450, CD83 APC, CD86 FITC and HLA-DR V500 (BD Bioscience). At least 10,000 CD11c+ cells of each condition were acquired on a FACS Canto II flow cytometer (BD Biosciences).
Quirant-Sánchez B., Mansilla M.J., Navarro-Barriuso J., Presas-Rodríguez S., Teniente-Serra A., Fondelli F., Ramo-Tello C, & Martínez-Cáceres E. (2021). Combined Therapy of Vitamin D3-Tolerogenic Dendritic Cells and Interferon-β in a Preclinical Model of Multiple Sclerosis. Biomedicines, 9(12), 1758.
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