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Clone mp6 xt22

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The Clone MP6-XT22 is a monoclonal antibody that recognizes the mouse CD40 ligand (CD40L) protein. It is commonly used in flow cytometry and cell-based assays to detect and quantify the expression of CD40L on the surface of various cell types.

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Lab products found in correlation

Clone pc61, by BioLegend (2 mentions) Clone xmg1, by BioLegend (2 mentions) Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions) Cholera toxin, by Merck Group (1 mentions) Ovalbumin (ova), by Merck Group (1 mentions) Rtk2758, by BioLegend (1 mentions) Cd4 clone gk1, by Thermo Fisher Scientific (1 mentions) Ifnγ clone xmg1, by BioLegend (1 mentions) Klrg1 clone 2f1, by BioLegend (1 mentions) Cd8 clone 53 6, by Thermo Fisher Scientific (1 mentions) Cd90 2 clone 53 2, by Thermo Fisher Scientific (1 mentions) Anti ifn γ apc, by BD (1 mentions) Anti il 2 pe, by BD (1 mentions) Fortessa x20, by BD (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Anti cd4 bv421, by BD (1 mentions) Anti cd8 bv711, by BioLegend (1 mentions) Brain heart infusion agar, by BD (1 mentions) Tissue tearor handheld homogenizer, by Biospec (1 mentions) Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)

Spelling variants of this product

MP6-XT22 (65 citations) TNFα (clone MP6-XT22; (8 citations) Anti-TNF-α (clone MP6-XT22, (7 citations) TNF (clone MP6-XT22), (4 citations) Anti-TNF (clone MP6-XT22, (3 citations) Anti-TNF-α-BV785 (clone MP6-XT22) (2 citations) αTNF-α (clone MP6-XT22; (2 citations)

10 protocols using clone mp6 xt22

1

Synthesis and Conjugation of PlGF-2 Peptide with Antibody

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The synthesis of PlGF-2123-144-α-TNF was performed as described previously [24 (link)]. Rat anti-mouse TNFα antibody (clone XT3.11, Bio X Cell, West Lebanon, NH, USA; or clone MP6-XT22, BioLegend, San Diego, CA, USA) was incubated with an excess amount of sulfo-SMCC or SM (PEG)4 crosslinker (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. Unreacted crosslinker was removed using a Zeba spin desalting column (Thermo Fisher Scientific), and then 15-fold molar excess of PlGF-2123-144 peptide (RRRPKGRGKRRREKQRPTDCHL) was added and reacted for 1 h at room temperature for conjugation to the thiol moiety on the C residue. The peptide had been synthesized with > 95% purity by Genscript (Piscataway, NJ, USA).
Katsumata K., Ishihara J., Fukunaga K., Ishihara A., Yuba E., Budina E, & Hubbell J.A. (2019). Conferring extracellular matrix affinity enhances local therapeutic efficacy of anti-TNF-α antibody in a murine model of rheumatoid arthritis. Arthritis Research & Therapy, 21, 298.
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2

Modulate Gut Immunity with Antibodies

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Six to eight-week-old mice were intra-peritoneally injected with anti-a4b7 IgG antibody (250μg in 250μl PBS) (Clone DATK32, Biolegend), anti-TNF IgG antibody (Clone MP6-XT22, Biolegend), isotype control IgG (RTK2758, Biolegend), or an equal amount of PBS.
For OVAlbumin (OVA) immunization, mice were sensitized with an intraperitoneal injection of 500μg OVA (Sigma Aldrich) and 0.1μg cholera toxin (CT) (Sigma Aldrich) on day 0, followed by oral administration of 10mg OVA and 10μg CT on day 7 and 14. On day 21. Stool and blood were collected to measure OVA-specific IgA and OVA-specific IgG, respectively.
Canales-Herrerias P., Uzzan M., Seki A., Czepielewski R.S., Verstockt B., Livanos A., Raso F., Dunn A., Dai D., Wang A., Al-taie Z., Martin J., Ko H.M., Tokuyama M., Tankelevich M., Meringer H., Cossarini F., Jha D., Krek A., Paulsen J.D., Nakadar M.Z., Wong J., Erlich E.C., Onufer E.J., Helmink B.A., Sharma K., Rosenstein A., Chung G., Dawson T., Juarez J., Yajnik V., Cerutti A., Faith J., Suarez-Farinas M., Argmann C., Petralia F., Randolph G.J., Polydorides A.D., Reboldi A., Colombel J.F, & Mehandru S. (2023). Gut-associated lymphoid tissue attrition associates with response to anti-α4β7 therapy in ulcerative colitis. bioRxiv.
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3

Comprehensive Immunophenotyping of T Cells

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The cells were stained with the following antibodies: CD8 (clone 53–6.7, eBioscience), CD4 (clone GK1.5, eBioscience), CD90.1 (clone OX-7, eBioscience), CD90.2 (clone 53–2.1, eBioscience), PD-1 (clone J43, Biolegend), KLRG1 (clone 2F1, Biolegend), p53 (clone pAb 240, Novus Biologicals) TNF (Clone MP6-XT22, Biolegend) and IFNγ (clone XMG1.2, Biolegend) at the appropriate dilution and with compatible fluorochromes. The lymphocyte gates in the samples were plotted directly to examine CD8 T cell populations depicted in the flow plots.
Kurup S.P., Moioffer S.J., Pewe L.L, & Harty J.T. (2020). P53 hinders CRISPR/Cas9 mediated targeted gene disruption in memory CD8 T cells in vivo. Journal of immunology (Baltimore, Md. : 1950), 205(8), 2222-2230.
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4

Analyzing Murine Typhimurium Infection

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Mice were allocated to control and experimental groups randomly, sample sizes were chosen on the basis of previous experience to obtain reproducible results, and the investigators were not blinded. Mice were inoculated intraperitoneally with 1 to 2 × 103 colony-forming units (CFU) S. Typhimurium SL1344 WT or ΔsteE in 200 μl of PBS. For TNF neutralization, infected mice were injected intraperitoneally with either 500 μg of anti-TNF monoclonal antibody, clone MP6-XT22 (BioLegend) or isotype control antibody in sterile PBS in 400-μl total volume before scRNA-seq or analysis 3 days later. Mice were euthanized at the indicated time points after inoculation by CO2 asphyxiation followed by cervical dislocation as the secondary method of euthanasia. Organs were collected, weighted, and either homogenized in PBS for CFU enumeration, used to make single-cell suspension for flow cytometric analysis, or prepared for microscopy examinations.
Pham T.H., Xue Y., Brewer S.M., Bernstein K.E., Quake S.R, & Monack D.M. (2023). Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection. Science Advances, 9(1), eadd4333.
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5

Cytokine profiling of CAR T cells

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CAR T cells and tumour cells were co-incubated for 5 h in T cell media at a 1:1 ratio in the presence of Golgi-stop (Becton Dickinson, Kit #554715, final concentration 5 ml/mL), at 37 °C, 5% CO2. Cells were subsequently incubated with antibodies to surface proteins in FACS buffer (Phosphate Buffered Saline (PBS) with 0.2% Bovine Serum Albumin (BSA, Gibco)), with anti-CD4-BV421 (Clone MP6-XT22, BD Horizon, Franklin Lakes, NJ, USA), anti-CD8-BV711 (Clone 53-6.7, BioLegend, San Diego, CA, USA), and the viability dye Fixable yellow (Invitrogen, Oregon) for 30 min at 4 °C. Cells were then fixed for 20 min at room temperature and permeabilised using BD Pharmingen Fix/Perm kit (Cat: 554715, BD Pharmigen, Franklin Lakes, NJ, USA) according to manufacturer’s instructions. Intracellular IFNγ, IL-2 and TNFα were detected by incubating with anti-IFNγ-APC (Clone XMG1.2, BD Pharmingen, Franklin Lakes, NJ, USA), anti-IL2-PE (Clone JES6-5H4; BD Biosciences, East Rutherford, NJ, USA), and anti-TNFα-AF488 (Clone MP6-XT22; Biolegend) antibodies for 1 h at 4 °C. Cells were washed with a FACS buffer and analysed using a Fortessa X20 (BD Biosciences, East Rutherford, NJ, USA) and analysis performed on FlowJo software V10.8 (TreeStar, Ashland, OR, USA).
Wang S.S., Luong K., Gracey F.M., Jabar S., McColl B., Cross R.S, & Jenkins M.R. (2021). A Novel Peptide-MHC Targeted Chimeric Antigen Receptor T Cell Forms a T Cell-like Immune Synapse. Biomedicines, 9(12), 1875.
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6

Listeria monocytogenes infection in immunized mice

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Immunized mice were challenged one week after the last boost with 105colony-forming units (CFU) of Listeria monocytogenes (Lm-GFP strain) (200
µL) by intraperitoneal injection. Negative controls included mice immunized with
sterile PBS and mice immunized with the nanofibers lacking a T-cell epitope. Mice
receiving a therapeutic TNF-neutralizing monoclonal antibody (500 µg, clone
MP6-XT22, Biolegend) 3 hours before Listeria monocytogenes challenge were
employed as the positive control. The spleen and liver were harvested 48h later. The
organs were placed in 5 mL sterile 0.05% Tween-20 in water, diced with scissors,
and homogenized with a Tissue Tearor hand held homogenizer (Biospec Products). The
homogenized sample (5 mL for the spleen or 6 mL for the liver) was serially diluted 3
times at 1:10 dilution in sterile 0.05% Tween-20 in water. 100 µL (spleen)
or 120 µL (liver) of undiluted, 10-, 100-, and 1000-fold diluted solutions were
plated on Brain-Heart Infusion Agar (BD Biosciences) in quadrant-divided Petri dishes. The
plates were allowed to air dry inside a sterile hood, then incubated upside down for up to
48 h in a 37°C incubator. The off-white Listeria colonies were
counted and the total CFU per organ were calculated using the formula: Total CFU= CFU
count × dilution factor × 50 (corrected for total organ homogenate
volume).
Mora-Solano C., Wen Y., Han H., Chen J., Chong A.S., Miller M.L., Pompano R.R, & Collier J.H. (2017). Active immunotherapy for TNF-mediated inflammation using self-assembled peptide nanofibers. Biomaterials, 149, 1-11.
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7

Multi-parameter T cell analysis

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Cells were stained with conjugated fluorescent monoclonal antibodies against CD69 (#104527, clone H1.2F3, BioLegend) and CD25 (#102024, clone PC61, BioLegend). After washing, cells were fixed and permeabilized with the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) as per the manufacturer’s instructions. Cells were stained with conjugated fluorescent monoclonal antibodies against IFNγ (#505807, clone XMG1.2, BioLegend), TNFα (#506303, clone MP6-XT22, BioLegend) and IL-2 (#503821, JES6-5H4, BioLegend). All samples were acquired on a Beckman Coulter Cytoflex LX flow cytometry system using single-color compensation controls to set gate margins and analyzed with FlowJo software (FlowJo LLC).
Sun Y., Revach O.Y., Anderson S., Kessler E.A., Wolfe C.H., Jenney A., Mills C.E., Robitschek E.J., Davis T.G., Kim S., Fu A., Ma X., Gwee J., Tiwari P., Du P.P., Sindurakar P., Tian J., Mehta A., Schneider A.M., Yizhak K., Sade-Feldman M., LaSalle T., Sharova T., Xie H., Liu S., Michaud W.A., Saad-Beretta R., Yates K.B., Iracheta-Vellve A., Spetz J.K., Qin X., Sarosiek K.A., Zhang G., Kim J.W., Su M.Y., Cicerchia A.M., Rasmussen M.Q., Klempner S.J., Juric D., Pai S.I., Miller D.M., Giobbie-Hurder A., Chen J.H., Pelka K., Frederick D.T., Stinson S., Ivanova E., Aref A.R., Paweletz C.P., Barbie D.A., Sen D.R., Fisher D.E., Corcoran R.B., Hacohen N., Sorger P.K., Flaherty K.T., Boland G.M., Manguso R.T, & Jenkins R.W. (2023). Targeting TBK1 to overcome resistance to cancer immunotherapy. Nature, 615(7950), 158-167.
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8

Isolation and Activation of Murine Splenocytes

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The spleen was harvested from a laboratory BALB/c mouse. After grinding and passing through the 200 meshes strainer, splenocytes were resuspended in RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamate, penicillin (100 U/mL), streptomycin (100 μg/mL), 2 μg/mL anti- mouse CD3 (Cat 100301, Clone 145-2C11, BioLegend), 1 μg/mL anti- mouse CD28 (Cat 102101, Clone 37.51, Biolegend), and 20 ng/mL recombinant mouse IL-2 (PeproTech). A total of 1 × 106 splenocytes per well were seeded into a 12-well plate, treated with 100 nM NP-IDO-APT, NP-Scr-APT, or NPs, and cultured for 6 days. For T cell function measurement, cells were stimulated with 100 ng/mL PMA, 500 ng/mL ionomycin, and protein transport inhibitor cocktail (eBioscience) for 5 h before harvest. Afterwards, cells were incubated with anti-CD45-PEcy7(Cat 103113, Clone 30-F11, Biolegend), anti-CD8-FITC (Cat 100705, Clone 53-6.7, Biolegend) for surface staining and incubated with anti-IFN-γ-APC (Cat 505809, Clone XMG1.2, Biolegend) or anti-TNF-α-PE (Cat 506305, Clone MP6-XT22, Biolegend) after fixation and permeabilization. For Treg cells analysis, cells were collected and stained with anti-CD4-PerCP (Cat 100537, Clone RM4-5, Biolegend), anti-CD25-PE (Cat 102007, Clone PC61, Biolegend), and anti-Foxp3-APC (Cat 77-5775-40, Clone FJK-16s, eBioscience). Subsequently, cells were measured by flow cytometry.
Zhu Z., Yang Z., Zhu C., Hu Z., Jiang Z., Gong J., Yuan Y., Chen X., Jin Y, & Yin Y. (2023). Development of a DNA aptamer targeting IDO1 with anti-tumor effects. iScience, 26(8), 107367.
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9

Assessing TNFα Neutralization in T-cell Inhibition

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To assess the role of the TNFα cytokine in growth inhibition of CD4 + T cells induced by the anti-Ly-6A antibody, the biological activity of TNFα was neutralized with an anti-TNFα antibody (Clone MP6-XT22) (BioLegend, San Diego, CA, USA). Cultures with isotype-matched antibody (BioLegend, San Diego, CA, USA) served as controls for determining the specificity in these blocking assays.
Patel A.G., Moxham S, & Bamezai A.K. (2023). Ly-6A-Induced Growth Inhibition and Cell Death in a Transformed CD4+ T Cell Line: Role of Tumor Necrosis Factor-α. Archivum immunologiae et therapiae experimentalis, 71(1).
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10

Murine Salmonella Infection Model

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Mice were allocated to control and experimental groups randomly,
sample sizes were chosen based on previous experience to obtain reproducible
results and the investigators were not blinded. For oral infections, food
was removed 16 hours prior to inoculation with 108 CFU
S. Typhimurium in 100 μL PBS by oral gavage. For
intraperitoneal infections, mice were injected with 103 CFU in
200 μL PBS. For TNF neutralization, infected mice were injected
i.p. with either 500 g anti-TNF monoclonal Ab, clone
MP6-XT22 (Biolegend), or isotype control Ab in sterile PBS in 400 L total
volume every 4 days until analysis. Mice were euthanized at the indicated
time points post-inoculation by CO2 asphyxiation followed by cervical
dislocation as the secondary method of euthanasia. Organs were collected,
weighted, and either homogenized in PBS for CFU enumeration, used to make
single cell suspension for flow cytometric analysis, or prepared or
histopathological examinations.
Pham T.H., Brewer S.M., Thurston T., Massis L.M., Honeycutt J., Lugo K., Jacobson A.R., Vilches-Moure J.G., Hamblin M., Helaine S, & Monack D.M. (2019). Salmonella-driven polarization of granuloma macrophages antagonizes TNF-mediated pathogen restriction during persistent infection. Cell host & microbe, 27(1), 54-67.e5.
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