Celastrol was obtained from Sigma-Aldrich (Saint Louis, Missouri), Zol from Novartis Pharmaceuticals (Basel, Switzerland), and rhIL-2 from Primegene Bio-Tech Co. (Shanghai, China), respectively. The following FITC- or PE –conjugated mAbs were obtained from eBioscience (San Diego, CA): anti-TCR-γδ, anti-CD3, anti-CD69, anti-DR4 (DJR1), and anti-DR5 (DJR2-4) mAbs were obtained from eBioscience (San Diego, CA). Additionally, the purified anti-human TRAIL-R and TRAIL were from BD Bioscience (San Diego, CA), Mouse monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich (Saint Louis, Missouri). Isotype-matched monoclonal antibodies were obtained from BD biosciences and eBioscience and used as staining controls. 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), RNase were purchased from Sigma (St. Louis, USA), Annexin-V/PI binding assay kit was from Invitrogen Ltd. (Paisley, UK), the CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega (Madison, WI), and IFNγ ELISA kits from Bender MedSystem (Vienna, Austria). Other chemicals were from Beyotime Biotechnology Company (Shanghai, China).
Isotype matched monoclonal antibody
Manufactured by BD
Sourced in United States
Isotype-matched monoclonal antibodies are laboratory reagents used to detect and quantify specific target molecules in biological samples. These antibodies are produced from a single clone of B cells and share the same antibody class (isotype). They are designed to provide a reliable and standardized tool for various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte, by Merck Group (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (1 mentions)
Celastrol, by Merck Group (1 mentions)
Pe conjugated mabs, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazolyl 2 2 5 diphenyltetrazolium bromide mtt rnase, by Merck Group (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cd90 pe, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Cd29 pe, by BD (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Peroxidase labelled secondary antibody, by Agilent Technologies (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
3 amino 9 ethylcarbazole plus substrate chromogen, by Agilent Technologies (1 mentions)
Phycoerythrin conjugated anti cd16, by BD (1 mentions)
Fluorescein isothiocyanate conjugated anti cd16, by BD (1 mentions)
6 protocols using isotype matched monoclonal antibody
1
Celastrol, Zol, and rhIL-2 Cytotoxicity Assay
2
Immunophenotyping analysis with monoclonal antibodies
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Anti-human monoclonal antibodies against CD3, CD4, CD8, CD56, CD19, CD45RO, CD62L, and CCR7, were used for immunophenotyping analysis. All these antibodies and isotype-matched monoclonal antibodies were purchased from BD Biosciences (CA, USA). Data acquisition was performed using a FACSCalibur flow cytometer (BD Biosciences).
3
Immunophenotyping of USC Cells
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Passage 4 USCs were incubated with 3 % bovine serum albumin for 30 minutes to block nonspecific antigens. The cells were then incubated with the following monoclonal antibodies (Becton Dickinson, USA): CD29-PE, CD73-PE, CD90-PE, CD44-FITC, CD45-FITC, CD34-APC and HLA-DR-PE. Nonspecific fluorescence was determined by incubation of similar cell aliquots with isotype-matched monoclonal antibodies (BD Biosciences). The cells were washed to remove unbound antibodies. Surface antigens were analyzed using a Guava easyCyte™ (Millipore, Billerica, MA, USA).
4
Flow Cytometry Surface Marker Profiling
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The protocol to characterize cell surface markers by flow cytometry was modified [44 (link)]. Briefly, cells were blocked with cold PBS containing 1% bovine serum albumin (BSA) for 30 min, then incubated with the following fluorescence-conjugated antibodies (Becton Dickinson, USA) for 1 h: CD29-PE, CD73-PE, CD90-PE, CD44-FITC, CD13-FITC, SSEA4-PE, CD31-FITC, CD45-FITC, CD34-PE and HLA-DR-PE. Isotype-matched monoclonal antibodies were used as controls (BD Biosciences). Cells were washed to remove unbound antibodies and analyzed by using Guava easyCyte™ (Millipore, Billerica, MA, USA).
5
Antigen-Specific T Cell Activation Assay
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Naïve CD4+CD25− T cells were isolated from the spleens of OT-II mice using a CD4+ CD25− T cell isolation kit (Miltenyi Botec Inc.). Both tDCs and cDCs were generated from BM cells of C57BL/6 mice, matured in the presence of 50 ng/mL IFN-γ and 50 ng/mL TNF-α for 24 h, and then pulsed with the OVA323–339 peptide (10 μg/ml) for 1 h. After washing with PBS, the DCs (2 × 104 cells/well) were co-cultured with purified OT-II CD4+ CD25− T cells (2 × 105 cells/well) for 4 days in a medium containing 100 U/ml of recombinant human IL-2 (PeproTech Inc.). In allogeneic MLR, the DCs (2 × 104 cells/well) generated from BM cells of C57BL/6 mice were cultured with naïve CD4+CD25− T cells (2 × 105 cells/well) isolated from the spleens of BALB/c mice for 4 days in a medium containing 10 U/ml of recombinant human IL-2 (PeproTech Inc.). In IL-10 blocking experiments, anti-IL-10 monoclonal antibody, or isotype-matched monoclonal antibody (10 μg/ml, both from BD Biosciences, San Jose, CA, USA) was added from the initiation of the culture.
6
Immunohistochemical Analysis of CD16 and OX40
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Standard indirect immunoperoxidase procedures were used for immunohistochemistry
(IHC; ABC-Elite, Vector Laboratories, Burlingame, California). Slides were
dewaxed and rehydrated in distilled water. Endogenous peroxidase activity was
blocked using 0.5% H2O2. Sections were incubated with 10% normal goat serum
(DakoCytomation, Carpinteria, California) for 20 minutes and incubated with
primary antibody at room temperature (RT). Primary antibodies used were specific
for CD16 and OX40 (polyclonal anti-CD134/OX40, ab119904, Abcam, Cambridge,
United Kingdom; biotinylated anti-CD16 were purchased from DAKO (Glostrup,
Denmark) and Novocastra (Newcastle, United Kingdom). Fluorescein
isothiocyanate-conjugated anti-CD16, phycoerythrin-conjugated anti-CD16,
Cy5-conjugated anti-CD16, and isotype-matched monoclonal antibody were purchased
from BD Bioscience (San Jose, California), as previously published by our group.22 (link),23 (link) Subsequently, sections were incubated with peroxidase-labelled secondary
antibody (DakoCytomation) for 30 minutes at RT. For antigen visualization,
sections were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen
(DakoCytomation) for 30 minutes and counterstained with Gill’s hematoxylin.
(IHC; ABC-Elite, Vector Laboratories, Burlingame, California). Slides were
dewaxed and rehydrated in distilled water. Endogenous peroxidase activity was
blocked using 0.5% H2O2. Sections were incubated with 10% normal goat serum
(DakoCytomation, Carpinteria, California) for 20 minutes and incubated with
primary antibody at room temperature (RT). Primary antibodies used were specific
for CD16 and OX40 (polyclonal anti-CD134/OX40, ab119904, Abcam, Cambridge,
United Kingdom; biotinylated anti-CD16 were purchased from DAKO (Glostrup,
Denmark) and Novocastra (Newcastle, United Kingdom). Fluorescein
isothiocyanate-conjugated anti-CD16, phycoerythrin-conjugated anti-CD16,
Cy5-conjugated anti-CD16, and isotype-matched monoclonal antibody were purchased
from BD Bioscience (San Jose, California), as previously published by our group.22 (link),23 (link) Subsequently, sections were incubated with peroxidase-labelled secondary
antibody (DakoCytomation) for 30 minutes at RT. For antigen visualization,
sections were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen
(DakoCytomation) for 30 minutes and counterstained with Gill’s hematoxylin.
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