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Meca 79

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MECA-79 is a laboratory equipment designed for the analysis of cell-mediated immunity. It is a tool used to measure and quantify the secretion of cytokines, such as interferon-gamma, from activated immune cells. The core function of MECA-79 is to provide researchers with a reliable method to assess cellular immune responses.

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Lab products found in correlation

Supersignal west pico or west dura chemiluminescent substrates, by Thermo Fisher Scientific (2 mentions) Chemidoc imaging system, by Bio-Rad (2 mentions) Image lab, by Bio-Rad (2 mentions) Nat105, by Abcam (1 mentions) Ep1150y, by Abcam (1 mentions) High or low ph antigen retrieval buffer, by Agilent Technologies (1 mentions) Epr1157 2, by Abcam (1 mentions) Envision flex system, by Agilent Technologies (1 mentions) Epr6855, by Abcam (1 mentions) Rat anti mouse monoclonal antibodies, by BD (1 mentions) Vectastain elite abc, by Vector Laboratories (1 mentions) Dab substrate kit, by Vector Laboratories (1 mentions) Fluorochrome conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) Mec13, by BD (1 mentions) Mca2077s, by Bio-Rad (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Bx51 microscope, by Olympus (1 mentions) Vectastain elite abc kit, by Vector Laboratories (1 mentions) Envision system, by Agilent Technologies (1 mentions)

Close spelling variants of this product

Anti-PNAd (MECA-79, (2 citations)

10 protocols using meca 79

1

Comprehensive Immunohistochemistry Profiling

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Tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Heat‐induced antigen retrieval was carried out in a high or low‐pH antigen retrieval buffer (DakoCytomation, Glostrup, Denmark). Endogenous peroxidase was blocked by incubating tissue sections in 3% H2O2 for 5 minutes. The primary antibodies against CD4 as a marker for helper T cells (1:500, EPR6855; Abcam, Cambridge, UK), CD8 as a maker for cytotoxic T lymphocytes (1:500, EP1150Y; Abcam), CD20 as a marker for pan B cells (1:50, L26; Abcam), Foxp3 as a marker for regulatory T cells (Treg) (1:300, 236A/E7; Abcam), PNAd as a marker for HEV (1:100, MECA‐79; BD PharmingenTM), programmed cell death (PD)‐1 as a marker for immune checkpoint molecules (1:50, NAT105; Abcam), Ki‐67 as a marker for cell proliferation (1:100, SP6; Abcam), CD80 as a marker for M1 macrophages (1:1000, EPR1157(2); Abcam) and CD163 as a marker for M2 macrophages (1:500, EPR11598; Abcam) were applied for 30 minutes. These sections were visualized using the HRP‐labeled polymer method (EnVision FLEX System, Dako). Immunostained sections were counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene. These data are summarized in Table S1.
Kuwabara S., Tsuchikawa T., Nakamura T., Hatanaka Y., Hatanaka K.C., Sasaki K., Ono M., Umemoto K., Suzuki T., Sato O., Hane Y., Nakanishi Y., Asano T., Ebihara Y., Kurashima Y., Noji T., Murakami S., Okamura K., Shichinohe T, & Hirano S. (2019). Prognostic relevance of tertiary lymphoid organs following neoadjuvant chemoradiotherapy in pancreatic ductal adenocarcinoma. Cancer Science, 110(6), 1853-1862.
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2

Tracking Wnk1-Dependent T Cell Trafficking

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CD44low T cells from Wnk1fl/+RCE and Wnk1fl/-RCE chimeric mice were labeled with CMAC or CMTMR for 15-20 min at 37°C, with dyes swapped between experiments. 3-4 x 106 cells were washed and injected intravenously at a 1:1 ratio into 5-10 week old C57BL/6J recipient mice. After 20 min, further adhesion of T cells to HEV was prevented by intravenous injection of anti-CD62L (Mel-14, 100 μg/mouse; Nanotools) in combination with Alexa Fluor 633-conjugated anti-PNAd (MECA-79, BD Biosciences, 15 µg/mouse) for visualization of HEVs by staining of PNAd. After a further 20 min (40 min after transfer) mice were sacrificed and perfused using 10 ml cold PBS and 10 ml cold 4% PFA. PLNs were processed as described3 (link). Images were visualized with Volocity software and T cells were counted manually and differentiated between cells in the lumen of HEV (“luminal”), attached to the outer MECA-79 signal (“perivascular”) and cells in the parenchyma of the peripheral lymph node (“parenchymal”). Alternatively mice were analyzed 20 min after transfer of T cells without injection of Mel-14.
Köchl R., Thelen F., Vanes L., Brazao T.F., Fountain K., Xie J., Huang C.L., Lyck R., Stein J.V, & Tybulewicz V.L. (2016). WNK1 kinase balances T cell adhesion versus migration in vivo. Nature immunology, 17(9), 1075-1083.
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3

Endometrial Glycans Expression Assay

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Purified mouse anti-blood group LewisY (LeY) monoclonal antibodies, clone LWY/1463, Abcam, ab 219,336, 1/200 (anti-LeY MAbs) and purified rat anti-mouse peripheral lymph node addressin (PNAd) carbohydrate epitope monoclonal antibodies, clone MECA-79, BD Pharmingen, Cat. No 553863, 1/100 (MECA-79 MAbs) were used to determine the expression of the glycans epitope in the endometrium. The anti-LeY MAbs recognize a difucosylated oligosaccharide LeY (major specificity Fucα1–2Galβ1–4(Fucα1–3)GlcNAcβ-R). MECA-79 MAbs react with carbohydrate epitopes which are a high-affinity L-selectin ligand (CD34, GlyCAM-1, MAdCAM-1). As previously reported, MECA-79 epitope contain (6-O-Su)GlcNAcβ-residue [23 (link)].
Ziganshina M.M., Dolgushina N.V., Kulikova G.V., Fayzullina N.M., Yarotskaya E.L., Khasbiullina N.R., Abdurakhmanova N.F., Asaturova A.V., Shchegolev A.I., Dovgan A.A, & Sukhikh G.T. (2021). Epithelial apical glycosylation changes associated with thin endometrium in women with infertility - a pilot observational study. Reproductive Biology and Endocrinology : RB&E, 19, 73.
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4

Quantifying Adoptive T Cell Localization

Cited 1 time
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For immunohistochemical analysis, serial 9 μm cryostat sections of TdLN or tumor were fixed in acetone, blocked with Superblock Blocking Buffer (Pierce), and stained with rat anti-mouse monoclonal antibodies specific for CD8α (53–6.7; BD Biosciences) [22 (link)]. Immune complexes were visualized using the Vectastain Elite ABC and DAB substrate kits (Vector Laboratories). For immunofluorescence histology, 9 μm cryosections were fixed at -20°C in methanol/acetone (3:1), and stained with monoclonal antibodies (anti-mouse PNAd antibody, MECA-79; anti-mouse CD31 antibody, MEC 13.3, BD Biosciences) and fluorochrome-conjugated secondary antibody (Jackson ImmunoResearch) as described [22 (link), 24 (link)]. Digital images of ≥10 randomly selected fields (unit area = 0.34 mm2 per field) were captured by observers blinded to sample identity using an Olympus BX50 upright fluorescence microscope equipped with a SPOT RT camera (Spectra Services). The number of adoptively transferred cells located within the parenchyma of TdLN or tumors (i.e., designated by location of fluorescent-labeled T cells outside PNAd+ or CD31+ vessels, respectively) was quantified with ImageJ software as described [21 (link), 24 (link), 25 ].
Ito F., Ku A.W., Bucsek M.J., Muhitch J.B., Vardam-Kaur T., Kim M., Fisher D.T., Camoriano M., Khoury T., Skitzki J.J., Gollnick S.O, & Evans S.S. (2015). Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer. PLoS ONE, 10(11), e0143370.
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5

Blood Group Antigen Detection

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SMSL and parotid saliva samples from 20 individuals were electrophoretically separated (4–12 % polyacrylamide gels) and transferred to nitrocellulose. The blots were probed with antibodies that recognize the A, B, Ley (Abcam), Lea, Leb (Neomarkers) blood group antigens and sulfated sialyl Lex (the L-selectin ligand, MECA-79, BD Biosciences). The method that we used has been published [49 ].
Albertolle M.E., Hassis M.E., Ng C.J., Cuison S., Williams K., Prakobphol A., Dykstra A.B., Hall S.C., Niles R.K., Ewa Witkowska H, & Fisher S.J. (2015). Mass spectrometry-based analyses showing the effects of secretor and blood group status on salivary N-glycosylation. Clinical Proteomics, 12, 29.
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6

Western Blot Analysis of PNAd

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Lymph nodes were lysed in ice-cold lysis buffer, and the protein concentration of cell lysates was measured using the Bradford (Bio-Rad) assay. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS)-0.05%Tween (TBST) and incubated overnight at 4°C with the following primary antibodies: MECA79 (anti-PNAd, BD Biosciences) and ERK1 (Santa Cruz Biotechnology). The blots were washed and developed with SuperSignal West Pico or West Dura chemiluminescent substrates (Thermo Scientific) using a Bio-Rad ChemiDoc imaging system, and the bands were quantified using Image Lab (Bio-Rad) software. MECA79 is a carbohydrate epitope found on a group of sulfation-decorated sialomucins, including sulfated ligands for CD62L (CD34, GlyCAM-1, Sgp200, and a subset of MAdCAM-1). This set of antigens has been referred to as PNAd with a molecular mass of 50–250 kD. Therefore, in the western blot, bands of multiple molecular weights were detected.
Azzi J., Yin Q., Uehara M., Ohori S., Tang L., Cai K., Ichimura T., McGrath M., Maarouf O., Kefaloyianni E., Loughhead S., Petr J., Sun Q., Kwon M., Tullius S., von Andrian U.H., Cheng J, & Abdi R. (2016). Targeted Delivery of Immunomodulators to Lymph Nodes. Cell reports, 15(6), 1202-1213.
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7

Tracking Wnk1-Dependent T Cell Trafficking

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CD44low T cells from Wnk1fl/+RCE and Wnk1fl/-RCE chimeric mice were labeled with CMAC or CMTMR for 15-20 min at 37°C, with dyes swapped between experiments. 3-4 x 106 cells were washed and injected intravenously at a 1:1 ratio into 5-10 week old C57BL/6J recipient mice. After 20 min, further adhesion of T cells to HEV was prevented by intravenous injection of anti-CD62L (Mel-14, 100 μg/mouse; Nanotools) in combination with Alexa Fluor 633-conjugated anti-PNAd (MECA-79, BD Biosciences, 15 µg/mouse) for visualization of HEVs by staining of PNAd. After a further 20 min (40 min after transfer) mice were sacrificed and perfused using 10 ml cold PBS and 10 ml cold 4% PFA. PLNs were processed as described3 (link). Images were visualized with Volocity software and T cells were counted manually and differentiated between cells in the lumen of HEV (“luminal”), attached to the outer MECA-79 signal (“perivascular”) and cells in the parenchyma of the peripheral lymph node (“parenchymal”). Alternatively mice were analyzed 20 min after transfer of T cells without injection of Mel-14.
Köchl R., Thelen F., Vanes L., Brazao T.F., Fountain K., Xie J., Huang C.L., Lyck R., Stein J.V, & Tybulewicz V.L. (2016). WNK1 kinase balances T cell adhesion versus migration in vivo. Nature immunology, 17(9), 1075-1083.
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8

Immunohistochemical Analysis of CYP19 and PNAd

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Collected tissues were fixed in 4% paraformaldehyde, embedded in paraffin wax, sectioned at 6 µm thickness, and stained with hematoxylin and eosin. Immunofluorescent labeling of CYP19 and PNAd were performed as previously described [24 (link)] by using monoclonal anti-CYP19 (MCA2077S; Bio-Rad Laboratories, USA) or purified rat anti-mouse PNAd carbohydrate epitope (MECA-79; BD Biosciences, USA). After primary antibody incubation overnight, slides were incubated with a secondary biotinylated horse anti-mouse IgG (Vectastain ABC kit; Vector Labs, USA) or rabbit anti-rat IgG (Vectastain ABC kit) and then treated with avidin-biotin complex solution (Vectastain Elite ABC kit; Vector Labs). For color development, 3,′-diaminobenzidine (DAB; Vector Labs) was applied. Slides were then counter-stained with hematoxylin, mounted, and imaged by using an Olympus BX51 microscope (Olympus, Japan).
Park C.J., Kim H., Jin J., Barakat R., Lin P.C., Choi J.M, & Ko C.J. (2018). Porcine intestinal lymphoid tissues synthesize estradiol. Journal of Veterinary Science, 19(4), 477-482.
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9

Western Blot Analysis of PNAd

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Lymph nodes were lysed in ice-cold lysis buffer, and the protein concentration of cell lysates was measured using the Bradford (Bio-Rad) assay. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS)-0.05%Tween (TBST) and incubated overnight at 4°C with the following primary antibodies: MECA79 (anti-PNAd, BD Biosciences) and ERK1 (Santa Cruz Biotechnology). The blots were washed and developed with SuperSignal West Pico or West Dura chemiluminescent substrates (Thermo Scientific) using a Bio-Rad ChemiDoc imaging system, and the bands were quantified using Image Lab (Bio-Rad) software. MECA79 is a carbohydrate epitope found on a group of sulfation-decorated sialomucins, including sulfated ligands for CD62L (CD34, GlyCAM-1, Sgp200, and a subset of MAdCAM-1). This set of antigens has been referred to as PNAd with a molecular mass of 50–250 kD. Therefore, in the western blot, bands of multiple molecular weights were detected.
Azzi J., Yin Q., Uehara M., Ohori S., Tang L., Cai K., Ichimura T., McGrath M., Maarouf O., Kefaloyianni E., Loughhead S., Petr J., Sun Q., Kwon M., Tullius S., von Andrian U.H., Cheng J, & Abdi R. (2016). Targeted Delivery of Immunomodulators to Lymph Nodes. Cell reports, 15(6), 1202-1213.
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10

Immunohistochemical Analysis of Vascular Markers

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The following monoclonal antibodies served as primary antibodies: QBEND10, which recognizes human CD34, a marker of vascular endothelial cells (mouse IgG; Immunotech, Luminy, France); MECA-79, which recognizes 6-sulfo N-acetyllactosamine attached to extended core 1 O-glycans (rat IgM; BD Pharmingen, San Diego, CA); 19, 24 and #15-8G-61, which recognizes human MAdCAM-1 (described above).
Immunohistochemistry for CD34 and MAdCAM-1 was undertaken using the EnVision system (Dako) and that for MECA-79 was carried out using an indirect method described previously. 31
Okamura T., Sakai Y., Hoshino H., Iwaya Y., Tanaka E, & Kobayashi M. (2015). Superficially located enlarged lymphoid follicles characterise nodular gastritis. Pathology, 47(1).
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