T2 cells were maintained in RPMI-IMDM medium containing 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY, USA) routinely in the lab. T2 cell-based in vitro binding affinity assay was performed as reported previously (15 (link)). Briefly, T2 cells (0.5 x 106/mL) was incubated with β2 microglobulin (3 μg/mL) (Sigma-Aldrich, St. Louis, MO,USA) and individual peptide (10 μg/mL) for 4 h at 37°C. L235 peptide was used as a positive control while RPMI-IMDM medium (GIBCO) as a negative control (15 (link)). After the incubation, flow cytometric analysis was performed to determine the expression of HLA-A2 on T2 cells. Briefly, cells were incubated with PE-mouse anti human-HLA-A2 antibody (Clone BB7.2) (Sigma-Aldrich) for 40 min at 4°C and acquired by FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was performed by using FlowJo software (Tree Star Inc., Ashland, OR, USA). Mean fluorescence index (MFI) was used to determine the expression level of HLA-A2 molecules on T2 cells. To determine the dissociation constant (Kd) value of each peptide, T2 cells were incubated with β2 microglobulin (3 μg/mL) and the peptides at different concentrations (0, 0.4, 2, 10, 20 μg/mL) followed by flow cytometric assay. Peptide concentration at 50% of maximum MFI represented the Kd values of each peptide.
β2 microglobulin
Manufactured by Merck Group
Sourced in United States
β2-microglobulin is a laboratory equipment product. It is a small protein found on the surface of most cells in the human body. β2-microglobulin is used in various medical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cfx manager software 3, by Bio-Rad (1 mentions)
Cfx real time system, by Bio-Rad (1 mentions)
Tnf α, by Merck Group (1 mentions)
Iscripttm cdna synthesis kit, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Beta actin antibody, by Proteintech (1 mentions)
Anti p62 antibody, by Cell Signaling Technology (1 mentions)
Sodium hydrosulfide, by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Goat anti mouse igg, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Citric acid, by Thermo Fisher Scientific (1 mentions)
N dodecyl β d maltoside, by Merck Group (1 mentions)
Milli q, by Merck Group (1 mentions)
α chymotrypsin, by Merck Group (1 mentions)
N n dimethyldodecylamine n oxide, by Merck Group (1 mentions)
Trizma hydrochloride, by Merck Group (1 mentions)
15 protocols using β2 microglobulin
1
HLA-A2 Binding Affinity Assay
2
Evaluating DEPDC1-derived Peptide Binding to HLA-A*0201
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Binding and stabilization of HLA-A*0201 molecule by DEPDC1-derived peptides was evaluated using T2 cells.57 (link) T2 cells were stripped in 0.131 M citric acid, 0.066 M Na2HPO4 (pH 3.3) for 45 s, washed and resuspended in serum-free culture media. A total of 2 × 105 cells were incubated with 3 μg/mL β2-microglobulin (Sigma-Aldrich) and 100 μg/mL peptide in a final volume of 500 μL for 4 h at 37 °C. Cells were then washed and stained with the FITC-conjugated HLA-A*0201 monoclonal antibody BB7.2 (BioLegend) before cytometry evaluation (FACSCalibur, BD Biosciences). Stabilization was calculated by dividing the Mean Fluorescence Intensity (MFI) of peptide-pulsed T2 cells with the MFI of T2 cells loaded with a negative control peptide (EYYELFVNI) with no predicted binding affinity to HLA-A*0201.58 (link)
3
Quantitative RT-PCR Analysis of Fibronectin Splicing
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Retrotranscription (RT) was performed in a final volume of 20 µl reaction using the iScriptTM cDNA Synthesis Kit according to the manufacturer’s instructions (Bio-Rad). For quantitative real-time PCR, RT samples were diluted up to 60 µl with bidistilled H2O, and 3 µl of the resulting cDNA was amplified in triplicate in 15 µl reaction mixture with 200 nM of each specific primer and SsoFast Evagreen Supermix (Bio-Rad) at 1× as final concentration. The amplification reaction was performed in a CFX Real-time system (Bio-Rad) as follows: 95°C for 5 min, followed by 35 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. Primers compassing the specific EIIIA exon of FN were as follows: forward, 5′-CCCTAAAGGACTGGCATTCA-3′; and reverse, 5′-CATCCTCAGGGCTCGAGTAG-3′. Pre-designated KiCqStart primers for human GAPDH and mouse IL-6, TNF-α, TLR2, TLR9, ITGA4, ITGA5, ITGA9, ITGAV, and β2-microglobulin genes were purchased from Sigma-Aldrich. The Bio-Rad CFX Manager software 3.0 was used for the normalization of the samples. β2-microglobulin gene expressions were used for the comparative concentration analysis. PCR splicing assays for human and mouse EDA, mouse EDB, and V region were performed as previously described (Malara et al., 2011 (link), 2016 (link)).
4
Peptide-HLA-A2 Complex Stability Assay
5
Sodium Hydrosulfide and β2-Microglobulin Assay
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Sodium hydrosulfide (NaHS, a donor of H2S) and β2-microglobulin were purchased from Sigma (Sigma, St. Louis, MO, United States). Anti-p62 antibody was purchased from Cell signaling Technology (Boston, MA, United States). Beta-actin antibody was purchased from Proteintech (Danvers, MA, United States).
6
Peptide-Induced HLA-A*0201 Upregulation
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The ability of the potential epitopes to bind to HLA-A*0201 molecules was investigated by detecting the up-regulation of peptide-induced HLA-A*0201 molecules on T2 cells. In summary, 1 × 106 T2 cells were cultured for 16 h at 37 °C and 5% CO2 with 50 μM of the generated peptides in serum-free RPMI 1640 media supplemented with β2- microglobulin (Sigma) at a concentration of 3 μg mL−1. Using the FACS Calibur flow cytometer (Becton Dickinson, USA), the expression of HLA-A*0201 on T2 cells was then assessed by labeling the cells with primary anti-HLA-A2 Ab obtained from BB7.2 and secondary antibody labeled with FITC (goat anti-mouse IgG, BD Biosciences Pharmingen, USA). Becton Dickinson, USA's Cell Quest software was used to evaluate the data. The Fluorescence index (FI) was calculated as follows: FI = (mean FITC fluorescence with the given peptide – mean FITC fluorescence without peptide)/(mean FITC fluorescence without peptide). After measuring the samples three times, the mean FI was determined. Positive and negative controls were HLA-A2.1-restricted MAGE-2 CTL epitope KMVELVHFL (amino acid position in MAGE-2; 112-120) and Kb-restricted Hpa CTL epitope FSYGFFVI (amino acid position in Hpa; 519-526).
7
Purification and Preparation of Biomolecules
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α-Chymotrypsin (from bovine pancreas, ≥85%, C4129), β2-microglobulin (from human urine, ≥98%, M4890), endothelin 1 (≥97%, E7764), endothelin 2 (≥97%, E9012), angiotensin I (≥90%, A9650), pentane (≥99%, 236,705), hexadecane (99%, H6703), Trizma hydrochloride (SLBG8541V), Trizma base (SLBK4455V), N,N-Dimethyldodecylamine N-oxide (LADO, ≥99%, 40,234), and n-Dodecyl β-D-maltoside (DDM, ≥98%, D4641) were obtained from Sigma-Aldrich. Human EGF (≥98%, CYT-217) was obtained from PROSPEC. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, 850,356P) and sphingomyelin (Brain, Porcine, 860,062) were purchased from Avanti Polar Lipids. Potassium chloride (≥99%, BCBL9989V) was bought from Fluka. Citric acid (≥99%, A0365028) was obtained from ACROS. All polypeptide biomarkers and chemicals were used without further purification. About 15 mM Tris base, pH 7.5 buffer used in this study was prepared by dissolving 1.902 g Trizma HCl and 0.354 g Trizma base in 1 l water (Milli-Q, Millipore, Inc.).
8
Peptide Binding Assay for HLA-A2
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10 mM peptide stocks were prepared in DMSO because of poor water solubility of the peptides and further diluted into complete IMDM. T2 cells, a kind gift from dr. Heemskerk (Leiden University Medical Centre, Leiden) were incubated with peptide concentrations ranging from 0.01–50 μM overnight and 3 μg/ml β-2 microglobulin (Sigma-Aldrich), and HLA-A2 expression was assessed with flow cytometry.
9
HLA-A2:Ig Dimer Staining for TCR Analysis
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For HLA-A2:immunoglobulin (Ig) dimer staining, Dimer-X reagent (BD Bioscience, San Diego, CA, USA) was prepared; 1 μg dimer, 5 μg LMP1166, and 0.5 μg β2-microglobulin (Sigma-Aldrich) at 37 °C overnight. TCR-transduced PBLs (1 × 106 cells) were incubated with LMP1166-loaded HLA-A2:Ig dimer for 40 min, and washed/stained with anti-human CD8a, CD4, and anti-mouse IgG1 for 20 min. For murine TCRs and in vivo persistence, 1 × 106 viable cells were stained with 0.5 μg indicated antibodies for 20 min. Fluorescence was measured using a FACS Calibur (BD Biosciences) and analyzed using FlowJo software (Tree Star, Otlen, Switzerland).
10
HLA-A*0201 Binding Assay with AKAP4 Peptides
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We used T2 cells to evaluate the binding and stabilization of HLA-A*0201 molecule stimulated by AKAP4-derived peptides. T2 cells were stripped in PBS for 2 h, washed, and resuspended in serum-free culture media. A total of 2 × 105 cells were incubated with 3 mg/ml β2-microglobulin (Sigma-Aldrich) and 50 μM peptide in a final volume of 200 μl for 4 h at 37°C. Cells were then washed and stained with the PE-conjugated HLA-A*0201 monoclonal antibody (BioLegend) before cytometry evaluation (FACSC II, BD Biosciences). As described elsewhere (19 (link)), fluorescence intensity (FI) was calculated as the mean fluorescence intensity (MFI) of peptide-pulsed T2 cells/the MFI of T2 cells not loaded with peptide − 1.
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