Cd14 v500 m5e2

Manufactured by BD

Sourced in United States

The CD14 V500 (M5E2) is a lab equipment product designed for cellular analysis. It provides reliable detection and quantification of the CD14 surface marker on cells. The product's core function is to facilitate the measurement and analysis of CD14-positive cells in a sample.

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Lab products found in correlation

Cd19 v500 hib19, by BD (2 mentions) Cd3viogreen bw264 56, by Miltenyi Biotec (1 mentions) Nkg2c apc, by Miltenyi Biotec (1 mentions) Nkg2a fitc, by Miltenyi Biotec (1 mentions) Macsquant cytometer, by Miltenyi Biotec (1 mentions) Cd56 apc vio770, by Miltenyi Biotec (1 mentions) Efluor 506, by Thermo Fisher Scientific (1 mentions) Pd l1 pe, by BioLegend (1 mentions) Cd4 apcefluor 780 rpa t4, by Thermo Fisher Scientific (1 mentions) Cd8 bv650 rpa t8, by BioLegend (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Live dead aqua, by Thermo Fisher Scientific (1 mentions) Cd4 pecy5 rpa t4, by BD (1 mentions) Pd 1 pe eh12.2h7, by BioLegend (1 mentions) Cd4 pe cy5, by BD (1 mentions) Cd19 v500, by BD (1 mentions) Ccr7 bv421, by BD (1 mentions) Icos pe, by BD (1 mentions) Cxcr5 af488, by BD (1 mentions) Pd 1 bv421, by BioLegend (1 mentions)

5 protocols using cd14 v500 m5e2

Comprehensive Phenotyping of NK Cells

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Frozen PBMCs from three AFB (CMV⁺) and six EUB (three CMV⁺, three CMV^-) donors were thawed and allowed to rest overnight, as previously described. For each donor, 10⁶ cells were resuspended in phosphate-buffered saline supplemented with 2% FBS and incubated with human Fc blocking solution (BD Biosciences) for 10 min at 4 °C. Cells were then stained with the following antibodies for 30 min at 4 °C: CD3 VioGreen (BW264/56, Miltenyi Biotec), CD14 V500 (M5E2, BD Biosciences), CD57 Pacific Blue (HNK-1, BioLegend), NKp46 PE (9E2/NKp46, BD Biosciences), CD16 PerCP-Cy5.5 (3G8, BD Biosciences), CD56 APC-Vio770 (REA196, Miltenyi Biotec), NKG2A FITC (REA110, Miltenyi Biotec) and NKG2C APC (REA205, Miltenyi Biotec). The cells were then washed and acquired on the MACSQuant cytometer (Miltenyi Biotec), and the data were analysed using FlowJo (v.10.7.1)⁷².

Aquino Y., Bisiaux A., Li Z., O’Neill M., Mendoza-Revilla J., Merkling S.H., Kerner G., Hasan M., Libri V., Bondet V., Smith N., de Cevins C., Ménager M., Luca F., Pique-Regi R., Barba-Spaeth G., Pietropaoli S., Schwartz O., Leroux-Roels G., Lee C.K., Leung K., Wu J.T., Peiris M., Bruzzone R., Abel L., Casanova J.L., Valkenburg S.A., Duffy D., Patin E., Rotival M, & Quintana-Murci L. (2023). Dissecting human population variation in single-cell responses to SARS-CoV-2. Nature, 621(7977), 120-128.

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Multiparametric Flow Cytometry Analysis of Activated CD4+ T Cells

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PBMCs were thawed and 1 million were added to a round-bottom 96-well plate and stimulated with the respective antigens (peptide pools, Mav and Mtb lysates, PHA and DMSO control). Plates were incubated at 37°C in a humidified CO₂ incubator for 20-24 hrs. Plates were centrifuged and cells were resuspended in PBS with 10% (v/v) FBS and incubated at 4°C for 10 min. Cells were then stained with fixable viability dye eFluor506 (eBioscience) and an antibody mixture containing anti-human CD3-AF700 (UCHT1; ThermoFisher), CD4-APCeFluor780 (RPA-T4; eBioscience), CD8-BV650 (RPA-T8; BioLegend), CD14-V500 (M5E2; BD Bioscience), CD19-V500 (HIB19; BD Bioscience), CD25-PerCPCy5.5 (BC96; BioLegend), CD69-PECy7 (FN50; eBioscience), CD137-APC (4B4-1; BioLegend), CD154-FITC (TRAP1; BD Bioscience), OX40-BV421 (Ber-ACT35; BioLegend), and PD-L1-PE (29E.2A3; BioLegend) for 30 min at 4°C. Cells were washed, resuspended in PBS and acquired on a BD LSR II flow cytometer (BD Biosciences). Analysis to compare frequencies of activated CD4+ T cells was completed on FlowJo. The total number of CD4+ T cells expressing combinations of activation markers was determined with background values (as determined from the medium alone control) subtracted. The gating strategy is found in Figure S1A.

Lindestam Arlehamn C.S., Benson B., Kuan R., Dill-McFarland K.A., Peterson G.J., Paul S., Nguyen F.K., Gilman R.H., Saito M., Taplitz R., Arentz M., Goss C.H., Aitken M.L., Horne D.J., Shah J.A., Sette A, & Hawn T.R. (2022). T-cell deficiency and hyperinflammatory monocyte responses associate with Mycobacterium avium complex lung disease. Frontiers in Immunology, 13, 1016038.

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Multiparameter Flow Cytometric Phenotyping

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Surface markers were evaluated using combinations of fluorochrome-conjugated monoclonal antibodies that were each titrated individually for their optimal stain index. PBMCs were stained at 10 million cells/mL in 200uL PBS. All PBMCs were incubated for 10 minutes with an amine-reactive viability dye (LIVE/DEAD Aqua, Invitrogen), washed twice, and then stained for 15 minutes at room temperature with combinations of monoclonal antibodies. For ex vivo phenotyping, cells were stained with CD3-AF700 (UCHT1, BD), CD4-PECy5 (RPA-T4, BD), CD8-APC-AF750 (3B5, Invitrogen), CD45RO-PETR (UCHL1, Beckman Coulter), CCR7-BV421 (150503, BD), CXCR5-AF488 (RF8B2, BD), PD-1-PE (EH12.2H7, BioLegend), CD14-V500 (M5E2, BD), and CD19-V500 (HIB19, BD). In vitro phenotyping was performed with combinations of CD3-AF700, CD4-PECy5, CD8-APC-AF750, CD45RO-PETR, CXCR5-AF488, CD14-V500, CD19-V500, ICOS-PE (DX29, BD), CD40L-PE (TRAP1, BD) and PD-1-BV421 (EH12.2H7, BioLegend). All PBMCs were washed twice after staining, fixed with 2% paraformaldehyde, and analyzed on a BD LSR Fortessa (BD Biosciences) at the VMC Flow Cytometry Shared Resource.
Flow cytometry data was analyzed using BD Biosciences FACSDiva Software. In all experiments, forward and side scatter were used to identify lymphocytes and from that population non-viable, CD14+, CD19+, CD8+ cells were excluded from further analysis (Fig. S1).

Nicholas K.J., Flaherty D.K., Smith R.M., Sather D.N, & Kalams S.A. (2017). Chronic HIV-1 infection impairs superantigen-induced activation of peripheral CD4+CXCR5+PD-1+ cells, with relative preservation of recall antigen-specific responses. Journal of acquired immune deficiency syndromes (1999), 74(1), 72-80.

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PBMC Isolation, Sorting, and RNA-Seq

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Cryopreserved PBMC were thawed in media containing 20% fetal bovine serum. Cell counts and viabilities were assessed using Guava ViaCount reagent and a Guava PCA (Millipore Sigma). Cells were washed and stained with Aqua Live/Dead stain (Molecular Probes), washed, and blocked using normal mouse IgG (Caltag). The cells were surface stained for CD3 FITC (clone UCHT1, Becton Dickinson [BD]), CD8 PerCP-eF710 (SK1, eBiosciences), CD14 V500 (M5E2, BD), CD45RO eF650NC (UCHL1, eBiosciences), CD4 APC (RPAT4, BD), CD45RA APC-H7 (HI100, BD), CD19 PE-Cy5 (SJ25-C1, Invitrogen), and CD56 PE-Cy7 (NCAM16.2, BD) (Figure S2). The cells were then washed and sorted/analyzed on the BD FACSAria II SORP Cell Sorter. Total RNA from sorted cell subsets was extracted using the Single Cell RNA Purification kit (Norgen Biotek Corp). RNA was prepared for NGS RNA sequencing (RNAseq) using the SMART-Seq technology as described previously (Ehrenberg et al., 2019 ; Picelli et al., 2014 (link); Ramsköld et al., 2012 (link); Shangguan et al., 2021 ). Briefly, cDNA was generated per manufacturer’s instructions from 2.5 ng of RNA, final exogenous ERCC RNA spike-in dilutions of 1:190,000, and 11 cycles of library PCR amplification. A total of 300 ng of cDNA was input template for final library processing using the Nextera XT kit (Illumina) per manufacturer’s instructions.

Li S.S., Hickey A., Shangguan S., Ehrenberg P.K., Geretz A., Butler L., Kundu G., Apps R., Creegan M., Clifford R.J., Pinyakorn S., Eller L.A., Luechai P., Gilbert P.B., Holtz T.H., Chitwarakorn A., Sacdalan C., Kroon E., Phanuphak N., de Souza M., Ananworanich J., O’Connell R.J., Robb M.L., Michael N.L., Vasan S, & Thomas R. (2022). HLA-B*46 associates with rapid HIV disease progression in Asian cohorts and prominent differences in NK cell phenotype. Cell host & microbe, 30(8), 1173-1185.e8.

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Multiparametric Flow Cytometry Analysis

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The following conjugated Abs were used in the present study: CD3-Alexa Fluor 700 (OKT3), CD8-BV650 (RPA-T8) (Biolegend, San Diego, CA, USA), CD4-APC-efluor 780 (RPA-T4), IFNγ-FITC (4S.B3) (Thermo Fisher Scientific, Waltham, MA, USA), CD14-V500 (M5E2), and CD19-V500 (HIB19) (BD Biosciences; San Jose, CA, USA). For the exclusion of dead cells, the Fixable Viability Dye eFluor™ 506 (LIVE/DEAD) was used (Thermo Fisher Scientific). To identify the IFNγ production on antigen-specific CD4⁺ T cells, one million of PBMCs were cultured with CD4⁺ YFV-MP (1 μg/mL), Dimethyl sulfoxide (DMSO) (1%) as a negative control, and Phorbol 12-Myristate 13-Acetate (PMA) (0.1 μg/mL) and ionomycin (1 μg/mL) as a positive control in the presence of brefeldin A (1 μg/mL) (BD Biosciences) for 6 h and subsequently permeabilized and stained as described previously [35 (link)]. Cells from donors have been excluded from the analysis if the IFNγ response to PMA and ionomycin stimulation was lower than 1% in the CD3⁺ cells. Cells were acquired on an LSR-II flow cytometer (BD Biosciences), and the analysis was performed using FlowJo 10.5.3. software (BD Biosciences).

Mateus J., Grifoni A., Voic H., Angelo M.A., Phillips E., Mallal S., Sidney J., Sette A, & Weiskopf D. (2020). Identification of Novel Yellow Fever Class II Epitopes in YF-17D Vaccinees. Viruses, 12(11), 1300.

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