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Af 410 na

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AF-410-NA is a laboratory instrument used for the detection and quantification of specific biological molecules. It functions as an enzyme-linked immunosorbent assay (ELISA) analyzer, capable of performing automated immunoassay measurements.

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Lab products found in correlation

Ab 108 c, by R&D Systems (3 mentions) Mab005, by R&D Systems (2 mentions) Mab406, by R&D Systems (2 mentions) Baf410, by R&D Systems (2 mentions) Horseradish peroxidase avidin, by R&D Systems (1 mentions) Elisa microplate reader, by Molecular Devices (1 mentions) Mp5 20f3, by BioLegend (1 mentions) Mp5 32c11, by BioLegend (1 mentions) Mlb00c, by R&D Systems (1 mentions) 410 mt cf, by R&D Systems (1 mentions) Precisionglide needle, by BD (1 mentions) Microliter syringe, by Hamilton Company (1 mentions) Il 18, by R&D Systems (1 mentions) Af 501 na, by R&D Systems (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Recombinant tnf α, by R&D Systems (1 mentions) Z vad fmk, by Promega (1 mentions)

10 protocols using af 410 na

1

Isolation and Stimulation of Primary Bone Marrow-Derived Macrophages

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Primary bone marrow derived macrophage (BMDM) was prepared by flushing the bone marrow of the tibia and femur of 6–8 week old C57BL/6J mice. The cells were grown in DMEM supplemented with 15% L929-conditioned media, 10% FBS and 1% penicillin/streptomycin for 7 days. BMDM were stimulated 250 µM palmitate conjugated with BSA or BSA as a control for 24 hours, or TNFα (10 ng/ml, 410-MT-010; R&D systems), IL-6 (40 ng/ml, 406-ML-005; R&D systems) and IL-4 (10 ng/ml, 404-ML-010; R&D systems) for 24 h. In some assays, differentiated BMDM were treated with conditioned media from 3T3-L1 adipocytes differentiated as described58 (link) and treated with 250 µM palmitate conjugated with BSA or BSA for 24 hours. In certain experiments, anti-TNFα (0.4 µg/ml, AF-410-NA; R&D systems) or anti-IL-6 (0.025 µg/ml, MAB406; R&D systems) blocking antibody or the same concentrations of IgG control (Normal Goat IgG: AB-108-C, Rat IgG1: MAB005; R&D systems) was added to conditioned media. Peritoneal macrophages were isolated as described33 (link). Mice were injected intraperitoneally with 1 ml of 3% (wt/vol) thioglycolate to elicit sterile peritonitis with macrophage numbers peaking on day 4. Cells were collected by lavage and used for migration assays.
Ramkhelawon B., Hennessy E.J., Ménager M., Ray T.D., Sheedy F.J., Hutchison S., Wanschel A., Oldebeken S., Geoffrion M., Spiro W., Miller G., McPherson R., Rayner K.J, & Moore K.J. (2014). Netrin-1 promotes adipose tissue macrophage accumulation and insulin resistance in obesity. Nature medicine, 20(4), 377-384.
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2

Kaempferol Modulates T-cell Cytokines

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The examine the effect of kaempferol on the IL-17, IL-21, and TNF-α production in differentiated T-cells, culture supernatants were obtained from Th17-polarized differentiated T-cells by the treatment of a indicated dose of kaempferol. The levels of IL-17, IL-21, and TNF-α in the supernatants of murine splenocyte cultures were measured sandwich enzyme-linked immunosorbent assay (Cat#:MAB721, Cat#:841338, AF-410-NA, R&D Systems, respectively). Horseradish peroxidase-avidin (R&D Systems) was used for color development Absorbance was measured at 405 nm on an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Lee C.J., Moon S.J., Jeong J.H., Lee S., Lee M.H., Yoo S.M., Lee H.S., Kang H.C., Lee J.Y., Lee W.S., Lee H.J., Kim E.K., Jhun J.Y., Cho M.L., Min J.K, & Cho Y.Y. (2018). Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis. Cell Death & Disease, 9(3), 401.
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3

ELISA for Mouse IL-6 and TNF-α

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ELISA analyses for IL-6 and TNF-α were performed as previously described [25 (link)]. Antibodies used were: anti-mouse IL-6 (MP5-20 F3, Biolegend) 0.5 μg/mL; biotin anti-mouse IL-6 (MP5-32C11, Biolegend) 1 μg/mL; anti-mouse TNF-α (R&D systems, AF-410-NA) 0.5 μg/mL; biotin anti-mouse TNF-α (R&D systems, BAF410) 0.25 μg/mL.
Chuang T.Y., Guo Y., Seki S.M., Rosen A.M., Johanson D.M., Mandell J.W., Lucchinetti C.F, & Gaultier A. (2016). LRP1 expression in microglia is protective during CNS autoimmunity. Acta Neuropathologica Communications, 4, 68.
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4

Quantifying mouse cytokines

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Supernatants from cell culture or serum were assayed for mouse TNF-α (AF-410-NA, R&D Systems) and IL-1β (MLB00C, R&D Systems) levels according to the manufacturer’s instructions.
Kong X., Liao Y., Zhou L., Zhang Y., Cheng J., Yuan Z, & Wang S. (2020). Hematopoietic Cell Kinase (HCK) Is Essential for NLRP3 Inflammasome Activation and Lipopolysaccharide-Induced Inflammatory Response In Vivo. Frontiers in Pharmacology, 11, 581011.
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5

TNF-α Depletion and Combination Therapy

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Procedures similar to those described for the anti-IL-1β antibody or recombinant IL-1β were used. To deplete TNF-α, mice were injected with 50 μg of a TNF-α-specific antibody (AF410-NA; R&D Systems) 1 day before infection by bacteria and then twice per week for 2 weeks thereafter. The Salmonellae and TNF-α combination therapy groups received an intratumoral injection of recombinant TNF-α (410-MT/CF; 0.25 μg in PBS; R&D Systems) every 2 days starting at 5 dpi and continuing until 11 dpi. Recombinant TNF-α injections were performed using a Microliter syringe (Hamilton Company) fitted with a PrecisionGlide Needle (BDM011455-1; BD Biosciences).
Kim J.E., Phan T.X., Nguyen V.H., Dinh-Vu H.V., Zheng J.H., Yun M., Park S.G., Hong Y., Choy H.E., Szardenings M., Hwang W., Park J.A., Park S., Im S.H, & Min J.J. (2015). Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1β. Theranostics, 5(12), 1328-1342.
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6

Macrophage-NK Cell Interaction in Aspergillus Infection

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Macrophages were collected following digestion by 0.2% EDTA at 37°C for 5 min and were reseeded into 12-well plates at a density of 0.2×106 cells/well following three washes with warm PBS buffer. An equal quantity of autologous purified NK cells was added to the plates, which were then were mixed softly. Swelling conidia were added to the macrophage/NK cell co-culture plates, with a ratio of macrophages to conidia of 1:5. To investigate the contact dependence of the interaction, macrophages and NK cells were separated by a membrane (0.4-µm pore size) in Transwell plates (cat. no. CLS3464-48EA; Costar; Corning Inc.). The swelling conidia were able to contact the macrophages but not the NK cells. Macrophages with conidia or NK cells with conidia were used as controls. The cells and culture supernatant were collected for subsequent experiments following co-culture at 37°C for 24 h. In the antibody neutralization test, macrophages were stimulated by A. fumigatus conidia (1:5) for 24 h and then co-cultured with the NK cells from the same individual for a further 12 h, in the presence or absence of Galectin-9 (cat. no. AF2045; R&D Systems, Inc.)-, IL-18 (cat. no. D044-3; R&D Systems, Inc.)- or TNF-α (cat. no. AF-410-NA; R&D Systems, Inc.)-specific antibodies when NK cells were added. The antibody working concentrations were all 10 µg/ml.
Zhang X., He D., Gao S., Wei Y, & Wang L. (2019). Aspergillus fumigatus enhances human NK cell activity by regulating M1 macrophage polarization. Molecular Medicine Reports, 20(2), 1241-1249.
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7

Cytokine Detection by ELISA

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ELISA for IL-6 and TNF-α were performed as previously described (25 (link)). Antibodies used were: anti-mouse IL-6 MP5–20F3 (Biolegend, 504501) 0.5μg/mL; biotin anti-mouse IL-6 MP5–32C11 (Biolegend, 504601) 1μg/mL; anti-mouse TNF-α (R&D systems, AF-410-NA) 0.5μg/mL; biotin anti-mouse TNF-α (R&D systems, BAF410) 0.25μg/mL.
Rosen D.A., Seki S.M., Fernández-Castañeda A., Beiter R.M., Eccles J.D., Woodfolk J.A, & Gaultier A. (2019). Modulation of the Sigma-1 Receptor-IRE1 pathway is beneficial in preclinical models of inflammation and sepsis.. Science translational medicine, 11(478), eaau5266.
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8

Immunofluorescence Labeling of Immediate Early Genes and Cytokines

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After light stimulation, networks were immediately fixed with 3.7% paraformaldehyde (PFA) for 20 min at room temperature. Samples were washed thrice with PBS/50mM glycine solution to neutralize PFA, and then with Tween-20 to make cell membranes permeable. A blocking solution made of 3% BSA, 5% goat serum and 50 mM glycine in PBS was used to minimize nonspecific labeling with antibodies. Cells were first incubated with primary antibody [c-fos: sc-253 (Santa Cruz Biotechnology, Dallas, TX); interleukin-1 beta (IL1): AF-501-NA, TNF: AF-410-NA (R&D Systems, Minneapolis, MN)] at room temperature for 1–2 h, washed thrice, and then treated with a suitable Alexa-633 labeled secondary antibody (Invitrogen) for 1–2 h. Samples were washed twice, labelled with 4′,6-diamidino-2-phenylindole (DAPI), and then mounted on glass slides using ProLong Gold anti-fade reagent (Invitrogen) following the manufacturer’s protocol. All samples were imaged within three days of preparation.
Jewett K.A., Taishi P., Sengupta P., Roy S, & Davis C.J. (2015). Tumor necrosis factor enhances the sleep-like state and electrical stimulation induces a wake-like state in co-cultures of neurons and glia. The European journal of neuroscience, 42(4), 2078-2090.
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9

TNFα-Induced Cell Death Mechanisms

Cited 1 time
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Recombinant TNFα (R&D Systems) was used at various concentrations as indicated in the results section and figure legends. For TNFα neutralizing experiments, a goat IgG anti-TNFα neutralizing antibody (10.0 μg/ml, R&D Systems, AF-410-NA) 30 (link) was incubated with conditioned medium for 1 hour prior to treatment of cells along with control conditions using a goat IgG isotype control (10.0 μg/ml, R&D Systems, AB-108-C). Z-VAD.fmk (Promega; Madison, WI, USA) was used at a concentration of 40 μM.31 (link) Z-VAD.fmk was added to primary culture at the same time as TNFα and incubated with the ICC for a total of 24 hours. Necrostatin-1 (nec-1) in both active and inactive (Millipore) forms was used at a concentration of 30 μM.32 (link), 33 (link) Nec-1 was added to ICC primary cultures 1 hour before the addition of TNFα and incubated with ICC for a total of 24 hours.
Eisenman S.T., Gibbons S.J., Verhulst P.J., Cipriani G., Saur D, & Farrugia G. (2016). Tumor necrosis factor alpha derived from classically activated “M1” macrophages reduces interstitial cell of Cajal numbers. Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society, 29(4), 10.1111/nmo.12984.
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10

Isolation and Stimulation of Primary Bone Marrow-Derived Macrophages

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Primary bone marrow derived macrophage (BMDM) was prepared by flushing the bone marrow of the tibia and femur of 6–8 week old C57BL/6J mice. The cells were grown in DMEM supplemented with 15% L929-conditioned media, 10% FBS and 1% penicillin/streptomycin for 7 days. BMDM were stimulated 250 µM palmitate conjugated with BSA or BSA as a control for 24 hours, or TNFα (10 ng/ml, 410-MT-010; R&D systems), IL-6 (40 ng/ml, 406-ML-005; R&D systems) and IL-4 (10 ng/ml, 404-ML-010; R&D systems) for 24 h. In some assays, differentiated BMDM were treated with conditioned media from 3T3-L1 adipocytes differentiated as described58 (link) and treated with 250 µM palmitate conjugated with BSA or BSA for 24 hours. In certain experiments, anti-TNFα (0.4 µg/ml, AF-410-NA; R&D systems) or anti-IL-6 (0.025 µg/ml, MAB406; R&D systems) blocking antibody or the same concentrations of IgG control (Normal Goat IgG: AB-108-C, Rat IgG1: MAB005; R&D systems) was added to conditioned media. Peritoneal macrophages were isolated as described33 (link). Mice were injected intraperitoneally with 1 ml of 3% (wt/vol) thioglycolate to elicit sterile peritonitis with macrophage numbers peaking on day 4. Cells were collected by lavage and used for migration assays.
Ramkhelawon B., Hennessy E.J., Ménager M., Ray T.D., Sheedy F.J., Hutchison S., Wanschel A., Oldebeken S., Geoffrion M., Spiro W., Miller G., McPherson R., Rayner K.J, & Moore K.J. (2014). Netrin-1 promotes adipose tissue macrophage accumulation and insulin resistance in obesity. Nature medicine, 20(4), 377-384.
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