Serum CatB-specific IgG1 and IgG2c levels were assessed by ELISA as previously described [12 (link)]. Briefly, Immulon 2HB flat-bottom 96-well plates (Thermo Fisher) were coated overnight at 4ºC with rCatB (0.5 μg/mL) in 100 mM bicarbonate/carbonate buffer at pH 9.6 (50 μL/well). The plates were washed three times with PBS-Tween 20 (PBS-T: 0.05%; Fisher Scientific) and were blocked as above for 90 min. Serial serum dilutions in duplicate were incubated in the plates for 2 hours. Control (blank) wells were loaded with PBS-T. After washing three times with PBS-T, goat anti-mouse IgG1-horseradish peroxidase (HRP) (Southern Biotechnologies Associates, Birmingham, AL) and goat anti-mouse IgG2c-HRP (Southern Biotechnologies Associates) were added to the plates and incubated for 1 hour at 37°C. After a final washing step, TMB substrate (50 μL/well; Millipore, Billerica, MA) was used for detection followed by 0.5 M H2SO4 after 15 min (25 μl/well; Fisher Scientific). Optical density (OD) was measured at 450 nm with an EL800 microplate reader (BioTek Instruments Inc.). The results are expressed as the mean IgG1/IgG2c ratio of the endpoint titers ± standard error of the mean. Endpoint titers refer to the reciprocal of the highest dilution that gives a reading above the cut-off calculated as previously described [31 (link)].
Pbs tween 20
Manufactured by Thermo Fisher Scientific
Sourced in United States
PBS Tween-20 is a buffered saline solution containing the non-ionic detergent Tween-20. It is commonly used in various laboratory procedures, such as Western blotting, ELISA, and immunohistochemistry, to help solubilize and stabilize proteins, reduce non-specific interactions, and facilitate the washing of samples.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Immulon 2hb flat bottom 96 well plates, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
El800 microplate reader, by Agilent Technologies (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Cmra cell tracker dye, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Ab205270, by Abcam (1 mentions)
Ab90776, by Abcam (1 mentions)
A9205, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Peptivator sars cov 2 prot s, by Miltenyi Biotec (1 mentions)
5 protocols using pbs tween 20
1
Measuring Serum Cathepsin B Antibodies
2
Multiplex Neuroinflammation Profiling in Paired Samples
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Paired serum and CSF samples were measured using the V-Plex Neuroinflammation Panel 1 (Meso Scale Discovery (MSD), Rockville, MD, USA). The Panel consisting of 38 analytes was run according to product protocol. The 96-well plates pre-coated with capture antibodies were blocked with 5% MSD Blocker A Solution. Calibrator dilutions were prepared and samples were diluted as recommended for each kit with MSD Diluents. Samples and calibrators were then added to the plates and incubated at room temperature with shaking for 2 h. Plates were washed three times with a home-prepared solution of 10 × phosphatebuffered saline (PBS), pH 7.4 (Corning, VA, USA)-Tween 20 (Fisher Scientific, PA, USA).
Detection antibodies were mixed with MSD Diluents as indicated in the protocols of each kit and incubated at room temperature with shaking for 1-2 h. Plates were washed three times with the PBS-Tween 20 solution. MSD Read buffer was added and plates were read on an MSD instrument (SECTOR Imager 6000 reader). Data were generated and interpolated using MSD Discovery Workbench software.
Detection antibodies were mixed with MSD Diluents as indicated in the protocols of each kit and incubated at room temperature with shaking for 1-2 h. Plates were washed three times with the PBS-Tween 20 solution. MSD Read buffer was added and plates were read on an MSD instrument (SECTOR Imager 6000 reader). Data were generated and interpolated using MSD Discovery Workbench software.
3
Live Cell Tracking and Immunostaining
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CMRA cell tracker dye (ThermoFisher Scientific, MA, USA) was diluted to a concentration of 25 μM in cell media and added to the culture flask to enable live tracking of cells. The cells were incubated with the dye for 30 minutes, followed by replacing the media containing the CMRA stain with a culture medium.
For immunostaining, the cells were fixed with 4% paraformaldehyde (ThermoFisher Scientific, MA, USA) and permeabilized with 0.1% Triton X-100 (J63521, Alfa Aesar, MA, USA) for 15 minutes. The permeabilized cells were then blocked with 4% Bovine Serum Albumin (BSA) (VWR, PA, USA) for 30 minutes, and each step was followed by washing the cells with PBS Tween 20 (ThermoFisher Scientific, MA, USA).
For immunostaining, the cells were fixed with 4% paraformaldehyde (ThermoFisher Scientific, MA, USA) and permeabilized with 0.1% Triton X-100 (J63521, Alfa Aesar, MA, USA) for 15 minutes. The permeabilized cells were then blocked with 4% Bovine Serum Albumin (BSA) (VWR, PA, USA) for 30 minutes, and each step was followed by washing the cells with PBS Tween 20 (ThermoFisher Scientific, MA, USA).
4
PFA Fixation and Immunostaining for Alpha-Actinin and YAP1
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The PFA fixation and immunostaining processes were performed according to our previously reported methods [6 (link)]. After drug stimulation and washout, cells were fixed with 2% PFA for 15 min at 37 °C then washed twice with PBS. After blocking the cells with blocking buffer [1× PBS Tween-20 (28,352; Thermo Fisher Scientific) containing 1.5% (v/v) bovine serum albumin (A9205; Sigma-Aldrich)] for 30 min at room temperature (22–25 °C), the cells were incubated with a polyclonal antibody against rabbit alpha-actinin (AB90776; Abcam, Cambridge, UK), diluted with blocking buffer to a final concentration of 2 μg/mL, at 4 °C for 18 h. To analyze YAP1 nuclear localization, we used a rabbit monoclonal antibody against active YAP1 (AB205270; Abcam), diluted with blocking buffer to a final concentration of 0.2 μg/mL, at 4 °C for 18 h. After discarding the primary antibody solution, cells were washed twice with PBS and incubated with a secondary antibody solution, including goat anti-rabbit IgG (H + L) secondary antibody (A11034, Alexa Fluor 488, 1:400) and Hoechst 33342 (1:800; Thermo Fisher) at 4 °C for 1 h. Finally, cells were washed twice with PBS, and each well was filled with PBS before performing high-content analysis (HCA).
5
SARS-CoV-2 T Cell Response Evaluation
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Peripheral blood mononuclear cells (PBMC) were obtained by density gradient isolation with Lymphocyte Separation Medium reagent (Corning Life Sciences). Cells were maintained in RPMI 1640 (Corning Life Sciences) supplemented with 10% FBS (Gibco), 100 mg/mL streptomycin (Gibco), and 100U/mL penicillin (Gibco). Cells were stimulated with 15-mer overlapping peptide-pool covering immunodominant domain surface Spike-protein (PepTivator® SARS-CoV-2 Prot-S, Miltenyi Biotec) or with 10% DMSO for 6 hours at 37°C and 5% CO2 in the presence of Brefeldin A (Thermo-Scientific) during the last 4 hours of the assay. After stimulation, surface staining with anti-CD3-FITC (UCHT1), CD4-PE (OKT4) and CD8-PE/Cy7 (SK1) antibodies (Biolegend) was performed for 30 minutes at 4°C. After staining, they were fixed with 4% PFA for 30 min and permeabilized with 0.05% PBS Tween-20 (Thermo-Scientific) for 30 min at RT. Cells were stained with anti-IFNγ-APC antibody (B27) (Biolegend) for 30 min at RT. Staining was acquired on a FACSCalibur cytometer and analyzed with FlowJo v10 software (BD Life Sciences). Since the lower limit of detection for conventional flow cytometry techniques is ∼0.02 to 0.05%, we set 0.05% as the lower limit for considering the cellular response positive for both T-CD4 and T-CD8.
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