Clone m a251

Manufactured by BD

Sourced in United States

The Clone M-A251 is a laboratory equipment designed for cell line and hybridoma cloning. It provides a consistent and reliable method for single-cell isolation and expansion. The core function of this product is to facilitate the clonal selection and propagation of cell lines.

Automatically generated - may contain errors

Lab products found in correlation

10 protocols using clone m a251

Activation of Memory CD4+ T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

T cells were cultured in RPMI 1640 medium supplemented with 2 mM
glutamine, 1% (v/v) nonessential amino acids, 1% (v/v) sodium pyruvate,
penicillin (50 U/ml), streptomycin (50 μg/ml) (all from Invitrogen) and
5% heat-inactivated human serum (Swiss Red Cross). Sorted memory CD4⁺T cells were labelled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl
ester (CFSE, ThermoFisher) and cultured at a ratio of 2:1 with irradiated
autologous monocytes untreated or pre-pulsed for 2-3 h with a peptide pool
(15mers overlapping of 10) covering the entire sequence of the variable region
of the NZM heavy and light chains (NZM peptide pool, 3 μM per peptide,
produced by A&A Labs). After 12 days, cells were stained with antibodies
to CD25–PE (BD Biosciences, clone M-A251, cat. no. 555432) and
ICOS–Pacific Blue (Biolegend, clone C398.4A, cat. no. 313522).

Cassotta A., Mikol V., Bertrand T., Pouzieux S., Le Parc J., Ferrari P., Dumas J., Auer M., Deisenhammer F., Gastaldi M., Franciotta D., Silacci-Fregni C., Fernandez Rodriguez B., Giacchetto-Sasselli I., Foglierini M., Jarrossay D., Geiger R., Sallusto F., Lanzavecchia A, & Piccoli L. (2019). A single T cell epitope drives the neutralizing anti-drug antibody response to natalizumab in multiple sclerosis patients. Nature medicine, 25(9), 1402-1407.

+ Open protocol

+ Expand

Intracellular Cytokine and Treg Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols

After stimulation, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) then immunophenotyped for intracellular IFN-γ, IL-4 and IL-17A expression using the human TH1/TH2/TH17 phenotyping kit (BD Pharmingen) following the manufacturer’s protocol. For Treg analysis, naïve PBMCs were stained with anti-human FITC CD4 (0.6 μg/ml, clone SK3; BD Biosciences) and anti-human APC-Cy7 CD25 (2.5 μg/ml, clone M-A251; BD Biosciences) for 10 min at 4°C in the dark. After incubation, cells were fixed and permeabilized and then stained with anti-human APC FoxP3 (1:11, clone 3G3; Miltenyi Biotec) for 30 min at 4°C in the dark. Appropriate isotype controls were included for each sample.

Musuraca G., De Matteis S., Napolitano R., Papayannidis C., Guadagnuolo V., Fabbri F., Cangini D., Ceccolini M., Giannini M.B., Lucchesi A., Ronconi S., Mariotti P., Savini P., Tani M., Fattori P.P., Guidoboni M., Martinelli G., Zoli W., Amadori D, & Carloni S. (2015). IL-17/IL-10 double-producing T cells: new link between infections, immunosuppression and acute myeloid leukemia. Journal of Translational Medicine, 13, 229.

+ Open protocol

+ Expand

Immune Phenotyping of T Cells and Dendritic Cells in Kawasaki Disease

Check if the same lab product or an alternative is used in the 5 most similar protocols

Given the role of T cells and myeloid dendritic cells (DC) in the pro- and anti-inflammatory pathways in KD, we focused on immune phenotyping these cell populations. Myeloid DC populations were characterized and enumerated by flow cytometry using the following monoclonal antibodies (mAb) : anti-human CD11c-APC, mouse IgG1κ, clone B-ly6, anti-human CD11b-APC-Cyanin 7 (Cy7), mouse IgG1κ, clone ICRF44, anti-human CD14-phycoerythrin (PE) Cy7, mouse IgG2aκ, clone M5E2, anti-human CD86-fluorescein isothiocyanate (FITC), mouse IgG1κ, clone 2331 (FuN-1) (eBioscience).
T cell populations were characterized and enumerated by flow cytometry using the following mAb: CD25 BV421, mouse IgG1 k, clone M-A251 (BD Bioscience), anti-human CD4-percp-Cy5.5, mouse IgG1k, clone RPA-T4, anti-human CD8, Alexa Fluor 700, clone RPA-T8, mouse IgG1k, anti-human CD45RA APC, mouse IgG2b k, clone HI100, anti-human CD127 FITC, mouse IgG1 k, clone eBioRDR5, anti-human HLA-DR APC-H7 clone G46-6, mouse IgG2a k (eBioscience). Data were acquired with FACS ARIA II and analyzed using FACSDiva (BS Biosciences, San Jose, CA).

Tremoulet A.H., Jain S., Jone P.N., Best B.M., Duxbury E.H., Franco A., Printz B., Dominguez S.R., Heizer H., Anderson M.S., Glodé M.P., He F., Padilla R.L., Shimizu C., Bainto E., Pancheri J., Cohen H.J., Whitin J.C, & Burns J.C. (2019). Phase I/IIa Trial of Atorvastatin in Patients With Acute Kawasaki Disease With Coronary Artery Aneurysms. The Journal of pediatrics, 215, 107-117.e12.

+ Open protocol

+ Expand

Multiparametric Flow Cytometry of PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Freshly-isolated PBMCs (5×10⁵ cells) were surface stained with the viability dye, Aqua Amine Reactive Dye (Invitrogen), as well as monoclonal antibodies directed against CD3 (Clone SP 34-2, BD Biosciences), CD4 (NIH-Nonhuman Primate Reagent Resource Program), CD8 (Clone 3B5, Invitrogen), CD25 (Clone M-A251, BD Biosciences or Clone 4E3, Miltenyi), CD95 (Clone DX2, BD Biosciences) or CD45RA (Clone 2H4, Beckman Coulter), CD27 (Clone M-T271, BD Biosciences) or CD28 (Clone CD28.2, BD Biosciences), and CD127 (Clone hIL-7R-M21, BD Biosciences) for 20 minutes at room temperature, fixed and then permeabilized with Affymetrix FoxP3 Fix/Perm Buffers (Affymetrix) per the manufacturer’s instructions. Permeabilized cells were then stained intracellularly for Ki-67 (Clone B56, BD Biosciences) and FoxP3 (Clone PCH101, Affymetrix) for 30 minutes at 4°C, washed in PBS-2% FBS and analyzed by flow cytometry. A minimum of 150,000 events was acquired on a BD LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.).

Baker C.A., Swainson L., Lin D.L., Wong S., Hartigan-O’Connor D., Lifson J.D., Tarantal A.F, & McCune J.M. (2015). Exposure to SIV In Utero Results in Reduced Viral Loads and Altered Responsiveness to Postnatal Challenge. Science translational medicine, 7(300), 300ra125.

+ Open protocol

+ Expand

Comprehensive T-cell Suppression Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the suppression assay, the following antibodies were used: CD3 (Pacific Blue, clone UCHT1, BioLegend), CD8 (PE, clone RPA-T8,BDPharmingen) and CD69 (APC, clone FN50, BioLegend). The percent infection was assessed by quantifying GFP expression using the following formula:

[1 - (% GFP + CD 4 + T cells cultured with CD 8 + Tcells) / (% GFP + CD 4 + T cells without effectors) x 100 %] .

The following markers were used to assess cell death, immune activation, and exhaustion: CD3 (PE, clone UCHT1, BD Pharmingen), CD8 (APC-H7, clone SK1, BD Biosciences), CD69 (APC, clone FN50, BioLegend), 7AAD (Read in PerCP/Cy5.5, BD Pharmingen), Annexin V (Read in V450, BD Biosciences) and PD1 (PerCP/Cy5.5, clone EH12.2H7, BioLegend)CD25 (FITC, clone M-A251, BD Pharmingen), HLA-DR (PE, clone L243, Biolegend) and Ki67 (APC, clone Ki67, Biolegend). Proliferation was assessed using the CellTrace cell proliferation kit from Invitrogen, and read in the FITC channel.

Kwaa A.K., Goldsborough K., Walker-Sperling V.E., Pianowski L.F., Gama L, & Blankson J.N. (2017). The effect of Ingenol-B on the suppressive capacity of elite suppressor HIV-specific CD8+ T cells. PLoS ONE, 12(5), e0174516.

+ Open protocol

+ Expand

Multiparameter Analysis of T Cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols

For Tregs and effector-like T cells analysis, stimulated PBMCs were stained with CD4-FITC (0.6 μg/ml, clone SK3, BD Biosciences) and CD25-APC-Cy7 (2.5 μg/ml, clone M-A251, BD Biosciences) for 10 min at 4 °C in the dark. After incubation, cells were fixed, permeabilized and stained with FoxP3-APC (clone 3G3, Miltenyi Biotec) and with either Tbet-PE (clone REA102, Miltenyi Biotec) or GATA-3-PE (clone REA174, Miltenyi Biotec) or RORγt-PE (clone REA278, Miltenyi Biotec) for 30 min at 4 °C in the dark. Appropriate isotype controls were included for each sample.

De Matteis S., Molinari C., Abbati G., Rossi T., Napolitano R., Ghetti M., Di Rorà A.G., Musuraca G., Lucchesi A., Rigolin G.M., Cuneo A., Calistri D., Fattori P.P., Bonafè M, & Martinelli G. (2018). Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients. Journal of Translational Medicine, 16, 172.

+ Open protocol

+ Expand

Activation of Memory CD4+ T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Phenotypic Analysis of Human PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Peripheral blood mononuclear cells (PBMCs) were prepared by density gradient centrifugation method using Human Lymphocyte Separation Medium (Solarbio Life Science, Beijing, China; Catalog# P8610), and were kept in liquid nitrogen until use. The freezing medium for PBMCs was 90% fetal bovine serum supplemented with 10% dimethyl sulfoxide in a concentration of 10⁸ cells per vial. PBMCs were stained with allophycocyanin (APC) Mouse Anti-Human CD3 (BD Pharmingen, San Jose, CA, USA; Clone SP34-2; Catalog# 557597), peridinin-chlorophyll-protein complex (PerCP) Mouse Anti-Human CD4 (BD Pharmingen, San Jose, CA, USA; Clone L200; Catalog# 550631), fluorescein isothiocyanate (FITC) Mouse Anti-Human CD25 (BD Pharmingen, San Jose, CA, USA; Clone M-A251; Catalog# 555431), and phycoerythrin (PE) Mouse Anti-Human CD127 (BD Pharmingen, San Jose, CA, USA; Clone HIL-7R-M21; Catalog# 560822) for 30 minutes in the dark. Cells were analyzed by BD FACS Calibur Flow Cytometer (BD Bioscience, San Jose, CA, USA).

He W., Hao S., Dong X., Zhang D, & Jia Z. (2023). Circulating cytokine profile and modulation of regulatory T cells in chronic hepatitis B patients with type 2 diabetes mellitus. Biomolecules and Biomedicine, 23(1), 53-62.

+ Open protocol

+ Expand

Flow Cytometric Analyses of Leukocyte Surface Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

Surface markers of whole-blood leukocytes were determined by standard flow cytometric analyses using FACS Calibur and Cellquest software (BD).¹ Leukocytes were gated into lymphocytes, monocytes, and granulocytes by forward- and side-scatter analysis. Percent-positive cells were quantified via direct immunofluorescence staining using fluorescein isothiocyanate (FITC)-conjugated antibodies with phycoerythrin (PE)-conjugated antibodies. After binding of fluorescently labeled antibodies, expression densities of individual antigens were recorded. The expression density of the relevant antigens was calculated as the mean fluorescence intensity (MFI) according to the equation:
Subsequent monoclonal antibodies were determined separately on monocytes: antibodies directed against HLA-DR (clone L243, BD), CD83 (clone HB15a, Beckman Coulter), and CD123 (clone 9F5, BD). Lymphocytes were evaluated using antibodies directed against CD25 (clone M-A251, BD Biosciences) on its own and together with CD4 (cloneRPA-T4, BD Biosciences), CD2 (clone 39C1.5, Beckman Coulter) either together with CD80 (clone L307.4, Immunotech) or CD86 (clone B-T7; Diaclone). A mouse FITC-IgG1 antibody (clone X40) in conjunction with PE-conjugated IgG2a (clone X39, both from BD Biosciences) served as the isotype controls.

Bizjak D.A., Treff G., Zügel M., Schumann U., Winkert K., Schneider M., Abendroth D, & Steinacker J.M. (2021). Differences in Immune Response During Competition and Preparation Phase in Elite Rowers. Frontiers in Physiology, 12, 803863.

+ Open protocol

+ Expand

Quantification of Immune Cell Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols

Quantitation of CD4, CD8, and T regs was performed on 100 μL of whole blood as described before [46] using monoclonal antibodies toward CD4 (clone SK3), CD8 (clone RPA-T8), CD25 (clone M-A251, all from BD), and CD127 (clone eBioRDR5; eBiosciences). NK cells were identified using antibodies toward CD3 (clone SK7), CD16 (clone 3G8, all BD), and CD56 (clone N901, Beckman Coulter). Samples were incubated for 20 min. Thereafter, cells were treated with lysing solution (BD). Finally, cells were washed with FACS-buffer (PBS, 5% filtered fetal calf serum, 0.5% BSA, 0.07% NaN3), and analyzed using flow cytometry (FACS-Canto-II) and FACS-Diva-V6.1.3-software (BD). CD4 and CD8 T cells were quantified among lymphocytes, and T regs were quantified as CD127 low CD4 T cells expressing CD25. NK cells were quantified as CD3 negative/CD56 positive lymphocytes with CD16 positive/negative and CD56 bright/dim subsets. Gating strategies for the identification of cellular subpopulations are provided in Supporting information Fig. S2. Absolute lymphocyte numbers were calculated based on differential blood counts.

Hauser I.A., Marx S., Sommerer C., Suwelack B., Dragun D., Witzke O., Lehner F., Schiedel C., Porstner M., Thaiss F., Neudörfl C., Falk C.S., Nashan B, & Sester M. (2021). Effect of everolimus-based drug regimens on CMV-specific T-cell functionality after renal transplantation: 12-month ATHENA subcohort-study results. European journal of immunology, 51(4).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!