Epithelial organoids or fibroblasts derived from a total of 13 biopsies were prepared for FACS as described [6 (link)]. To isolate CD271 (nerve growth factor receptor)low/mucin 1 (MUC1)high, luminal epithelial cells, suspended cells from organoids were incubated for 30 minutes at 4 °C in the presence of CD271-APC (ME20.4, 1:50, Cedarlane Laboratories) and MUC1 (115D8, 1:50, Monosan) followed by AF488 (IgG2b, 1:500, Life Technologies). Fibroblasts were incubated with CD105-AF488 (SN6, 1:25, AbD Serotec) and CD26 (202–36, 1:200, Abcam), followed by AF647 (IgG2b, 1:500). Controls were without primary antibody. 1 μg/ml propidium iodide (Invitrogen) or Fixable Viability Stain 780 (1:1000, BD Biosciences) was added 10 minutes prior to analysis and sorting (FACSAria I and II; BD Biosciences).
Igg2b
Manufactured by Abcam
Sourced in United Kingdom
IgG2b is an antibody isotype that is part of the immunoglobulin G (IgG) class. It is a type of antibody molecule produced by B cells in response to an antigen. IgG2b antibodies play a role in the immune system's humoral response.
Automatically generated - may contain errors
Lab products found in correlation
Igg2a, by Abcam (9 mentions)
Dab chromogen reagent, by Vector Laboratories (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Fixable viability stain 780, by BD (1 mentions)
Igg2c, by Abcam (1 mentions)
Anti human fviii igg2a, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Elisa reader, by Tecan (1 mentions)
Spn c52h9, by ACROBiosystems (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Igg2c, by Southern Biotech (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by BD (1 mentions)
Cellquest, by BD (1 mentions)
Soluble tmb kit, by CWBIO (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Alum adjuvant, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
13 protocols using igg2b
1
Isolation of Luminal Epithelial Cells and Fibroblasts
2
Anti-FVIII IgG Subclass Quantification
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IgG subclass of anti-FVIII antibodies was assessed using an in-house established ELISA assay. Briefly, 96-well plates (Corning) were coated overnight with 1 µg/mL rhFVIII in PBS at room temperature. Plates were blocked using 1% bovine serum albumin (Sigma-Aldrich) in PBS. Standards or plasma samples were diluted in 1% bovine serum albumin in PBS. Anti-FVIII IgG isotype-specific antibodies were detected by anti-mouse horseradish peroxidase–conjugated IgG1, IgG2a (both from Southern Biotech, Birmingham, AL), IgG2b, or IgG2c (both from Abcam) and 3,3',5,5'-tetramethylbenzidine substrate solution (Perbio Science). Absorbance was detected at 450 nm using an ELISA reader (Tecan). The concentration of anti-FVIII isotype–specific antibodies was estimated from a standard curve obtained using serial dilutions of mouse anti-human FVIII IgG1 (Merck Millipore), anti-human FVIII IgG2a (Thermo Fisher Scientific) starting at 50 ng/mL, or mouse anti-FVIII reference plasma starting at 50 arbitrary units per milliliter.
3
SARS-CoV-2 Spike Protein Antibody Titers
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Anti-spike antibody titers were measured using ELISA as previously described19 (link). Briefly, 96-well plates were coated with 1 μg/ml recombinant spike protein (ancestral spike; SPN-C52H9, Omicron BA.1 spike; SPN-C52Hz, Omicron BA.2 spike; SPN-C5223, Omicron BA.5 spike; SPN-C522e, Acro Biosystems) and kept at 4 ℃ overnight. After blocking with 5% skim milk PBS for 2 h at RT, the diluted sera (ranging from 10- to 31,250-fold dilution with 5% skim milk PBS) were added to the wells and incubated at 4 °C overnight. The following day, the wells were washed with PBS-T and incubated with HRP-conjugated anti-IgG antibody (GE Healthcare) for 3 h at RT. The wells were developed using the peroxidase chromogenic substrate 3,3′-5,5′-tetramethyl benzidine (TMB, Sigma) for 30 min at RT. The reaction was quenched by adding sulfuric acid (0.5 N). The absorbance of the samples in the wells was measured immediately at 450 nm using a microplate reader (Bio-Rad). The half-maximum antibody titer of the sample was determined from the highest absorbance in the dilution range (GraphPad Prism 8 software). For IgG subclass determination, anti-IgG1 (Abcam), IgG2a (Abcam), IgG2b (Abcam), and IgG2c (Southern Biotech) antibodies were used as the secondary antibodies.
4
Enzyme-Linked Immunosorbent Assay for Influenza Antibodies
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Specific IgG and IgA titers were measured by enzyme linked immunosorbent assay (ELISA) using serum, BAL, or nasal samples from each mouse (n = 5), as previously described [37 (link)]. The plates were coated overnight at 4°C with the M2e peptides (5 μg/ml) or purified viruses (2 μg/ml) in PBS pH 7.2. The synthetic peptides used were M2es (SLLTEVET S E S from a consensus of human influenza A viruses). The purified viruses were A/Aichi/2/68(H3N2), A/California/07/09 (H1N1pdm09), A/California/1/66 (H2N2), A/Chicken/Kurgan/05/05 RG (H5N1), or A/Shanghai/2/2013(H7N9)-PR8-IDCDC H7N9. Polyclonal HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, IgG2b, or IgG3 antibodies (Abcam, UK) were used. To detect IgA, polyclonal HRP-labeled goat anti-mouse IgA (Abcam, UK) was used. TMB (BD Bioscience, USA) was used as substrate; the incubation time was 15 min. The optical density (OD) was measured using an i-Mark microplate reader (Bio-Rad) at a wavelength of 450 nm. The maximal serum dilution that had an optical density at least double the mean value of the blank was taken as the titer.
5
Immune Cell Surface Marker Profiling
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Treated and non-treated MΦ, were detached, and re-suspended in staining medium (DMEM with sodium azide). Immuno-labeling of bovine (preliminary analysis) and murine cell surface markers was performed by incubating cells with 15 μg/mL fluorescein isothiocyanate (FITC) conjugated antibodies to CD40 (Abcam), CD86 and CD80 (Thermo-Scientific) and isotype matched control mAbs IgG2a and IgG2b (Abcam) for 30 min on ice. After incubation, cells were washed three times with DMEM media containing 0.01% sodium azide and re-suspended in 400 μL 1X PBS (pH-7.4) containing 1 μg/mL propidium iodide, for excluding dead cells, and analyzed by flow cytometry with parameters set to 10,000 events, filter setting 530/30 nm wavelength (FACS Caliber) using acquisition software BD CellQuest (BD Biosciences) and the analysis program FlowJo 9.8.5 (TreeStar) at digital imaging core facility (College of Veterinary Medicine, Texas A&M University, College Station, TX).
6
LPS-specific Antibody Quantification
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96‐well plates were coated with LPSKpO2 extracted from K. pneumoniae stain 355 (10 µg well−1), incubated them at 4 °C overnight, washed them three times with wash buffer (PBS with 0.05% Tween 20) and dried them. Plates were blocked with blocking buffer (wash buffer with 5% skim milk powder; 200 µL well−1) at 37 °C for 2 h. After drying the plates, they were incubated in serially diluted serum (100 µL well−1) from each immunized mouse at 37 °C for 1 h. Next, plates were washed another three times and dried. Diluted HRP‐linked goat‐anti‐mouse antibodies (IgG, IgG1, IgG2a, IgG2b, and IgG3 [Abcam, Cambridge, UK]) (100 µL well−1) were added, and plates were incubated for another 1 h at 37 °C. After the washing step, a Soluble TMB Kit (CWBio, Beijing, China) was used to initiate a color‐producing reaction and measured the absorbance of each well at an OD of 450 nm.
7
Serum Antibody Detection for Truncated Tau
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Serum antibody specific for mis-disordered truncated tau (151–391/2N4R) was detected by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well plates (Dynex Technologies, Chantilly, VA, USA) were coated with 250 ng of recombinant full-length tau isoform 2N4R or pathological truncated tau (151–391/2N4R) per well at 4 °C overnight, then washed twice with PBS and blocked with 3% bovine serum albumin (in 0.05%PBS with Tween-20) for 2 hours at 37 °C. After blocking, the plates were incubated with serial dilutions of the serum (100 μl/well in twofold or fivefold dilution steps) for 1 hour at 37 °C. The bound serum antibodies were detected with horseradish peroxidase (HRP)-conjugated goat antimouse immunoglobulin G (IgG) (Zhongshan Golden Bridge Biotechnology Co., Beijing, China) and chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (Thermo Fisher Scientific).
To determine the isotypes of the specific antibodies produced in response to vaccine, mis-disordered truncated tau (151–391/2N4R) was coated onto ELISA plates, and sera from immunized mice were diluted at 1:6000 and added to the plate, followed by the addition of HRP-conjugated IgG1, IgG2a, IgG2b, and IgG3 (Abcam, Cambridge, UK).
To determine the isotypes of the specific antibodies produced in response to vaccine, mis-disordered truncated tau (151–391/2N4R) was coated onto ELISA plates, and sera from immunized mice were diluted at 1:6000 and added to the plate, followed by the addition of HRP-conjugated IgG1, IgG2a, IgG2b, and IgG3 (Abcam, Cambridge, UK).
8
Immunostaining for Amyloid Plaque Binding
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Example 5
Assessing Binding or Lack Thereof to Plaques/Fibrils
For immunostaining, antibodies described herein, positive control 6E10 (1 μg/ml) and isotype controls such as IgG1, IgG2a, and IgG 2b (1 μg/ml, Abcam) are used as primary antibodies. Sections are incubated overnight at 4° C., and washed 3×5 min in TBS-T. Anti-mouse IgG Horseradish Peroxidase conjugated (1:1000, ECL) is applied to sections and incubated 45 min, then washed 3×5 min in TBS-T. DAB chromogen reagent (Vector Laboratories, Burlington ON, Canada) is applied and sections rinsed with distilled water when the desired level of target to background staining is achieved. Sections are counterstained with Mayer's haematoxylin, dehydrated and cover slips were applied. Slides are examined under a light microscope (Zeiss Axiovert 200M, Carl Zeiss Canada, Toronto ON, Canada) and representative images captured at 50, 200 and 400× magnification using a Leica DC300 digital camera and software (Leica Microsystems Canada Inc., Richmond Hill, ON).
9
Mincle-targeted Cancer Immunotherapy
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DSPC, cholesterol and CP were purchased from Sigma Aldrich. Alum adjuvant was purchased from Thermo Fisher. Human Mincle-Fc and hMincle-his proteins were purchased from SinoBiological and Novoprotein, respectively. MCF-7, CT-26 and B16-F10 cancer cells were purchased from American Type Culture Collection (ATCC). Minimum Eagle's medium (MEM), RPMI medium 1640 and fetal bovine serum (FBS) were purchased from Gibico. Trypsin–EDTA was purchased from Invitrogen. HRP-linked goat anti-mouse kappa, IgM, IgG1, IgG2a, IgG2b, and IgG3 antibodies were purchased from Abcam. The FITC-labeled goat anti-mouse IgG antibody and LDH Cytotoxicity Detection Kit were purchased from Beyotime Biotechnology. Rabbit complements were purchased from Merck. Female BALB/c mice used for immunological studies were purchased from Southern Medical University (Guangzhou, China).
10
Immunostaining of Amyloid Plaques
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Example 7
Assessing Binding or Lack thereof to Plaques/Fibrils
For immunostaining, antibodies described herein, positive control 6E10 (1 μg/ml) and isotype control IgG1, IgG2a, IgG2b, or IgG3 (1 μg/ml, Abcam) are used as primary antibodies. Sections are incubated overnight at 4° C., and washed 3×5 min in TBS-T. Anti-Mouse IgG Horseradish Peroxidase conjugated (1:1000, ECL) is applied to sections and incubated 45 min, then washed 3×5 min in TBS-T. DAB chromogen reagent (Vector Laboratories, Burlington ON, Canada) is applied and sections rinsed with distilled water when the desired level of target to background staining is achieved. Sections are counterstained with Mayer's haematoxylin, dehydrated and cover slips were applied. Slides are examined under a light microscope (Zeiss Axiovert 200M, Carl Zeiss Canada, Toronto ON, Canada) and representative images captured at 50, 200 and 400× magnification using a Leica DC300 digital camera and software (Leica Microsystems Canada Inc., Richmond Hill, ON).
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