The immune response of Th cells to APCs pulsed with 10 μg/mL peptides or 50 μg/mL recombinant proteins was assessed by IFNγ ELISPOT assays (BD Biosciences) according to manufacturer’s instructions and as described previously.41 (link),45 (link) In brief, the frequency of peptide-specific CD4+ T-cells producing IFNγ was analyzed per 3 × 104 bulk CD4+ T-cells stimulated with equal numbers of peptide-pulsed autologous PBMCs, or alternatively, 1 × 104 bulk CD4+ T-cells stimulated with 5 × 104 peptide-pulsed L-cells expressing HLA-DR or -DP molecules To determine the HLA molecules involved in antigen presentation, antigen-induced IFNγ production was blocked by adding anti-HLA-DR monoclonal antibody (mAb; L243, IgG2a, BioLegend), anti-HLA-DP mAb (B7/21, IgG3, Abcam), anti-HLA-DQ mAb (SPV-L3, IgG2a, Abcam), or the broad spectrum anti-HLA class I mAb, (W6/32, IgG2a, Abcam). All mAbs were used at a final concentration of 5 μg/mL. Cells cultured with HIV-derived peptides were used as negative control. Cells cultured with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) were used as positive controls. All IFNγ ELISPOT assays were performed in triplicate or duplicate, and results are presented as means ± SD.
Igg2a
Manufactured by Abcam
Sourced in United Kingdom, United States
IgG2a is a type of antibody. It is a subclass of immunoglobulin G (IgG) antibodies. IgG2a plays a role in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Igg2b, by Abcam (9 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Dab chromogen reagent, by Vector Laboratories (2 mentions)
Ionomycin, by Merck Group (1 mentions)
Spv l3, by Abcam (1 mentions)
Ifnγ elispot assays, by BD (1 mentions)
Microplate reader, by Thermo Fisher Scientific (1 mentions)
96 microwell plates, by Corning (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Histostain sp kit, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Abcam (1 mentions)
Igg2c, by Southern Biotech (1 mentions)
Spn c52h9, by ACROBiosystems (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)
Nunc immuno maxisorp plates, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Tmb chromogen solution, by Thermo Fisher Scientific (1 mentions)
29 protocols using igg2a
1
Assessing Peptide-Specific CD4+ T-Cell Response
2
ELISA for Anti-p30 and Anti-p54 IgG
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Antibody responses to p30 and p54 were measured by indirect ELISA. Briefly, 96-microwell plates (Costar, Cambridge, MA, USA) were pre-coated with recombinant p30 (0.125 µg/mL) or modified p54 (0.5 ug/mL) at 100 µL/well in coating buffer (0.1 M carbonate buffer, pH 9.0). After incubation overnight at 4 °C, each plate was blocked with 5% skim milk (200 μL/well) for 2 h at 37 °C and washed five times with PBST. Then, serum samples (1:100 dilutions) from immunized mice were added at 100 μL/well and incubated for 1 h at 37 °C. Positive, negative and blank controls were also established. Subsequent steps were performed in accordance with a conventional protocol. The OD at 450 nm (OD450) of each well was detected using a microplate reader (Thermo Fisher Scientific). IgG1 and IgG2a were measured in the same way, except that serum samples were diluted in a twofold series and the secondary antibodies were HRP-labeled goat anti-mouse IgG1 or IgG2a (Abcam) instead of HRP-labeled goat anti-mouse IgG. IgG levels to p30 and p54 were expressed as OD450 values. To calculate titers of IgG1 and IgG2a, a cutoff value was defined as the mean specific OD450 value plus three standard deviations from mouse sera at 0 dpv, with a dilution of 1:100. The p30- and p54-specific IgG1 and IgG2a titers corresponded to the reciprocal values of the highest dilutions that showed positive.
3
Quantification of TIMP2 and MMP2 Expression
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Briefly, first, the samples were decalcified in 10% EDTA, and then embedded in paraffin. After the slices were created, the Histostain-SP Kit (Invitrogen) was used to evaluate the effects according to the manufacturer’s instructions. The primary antibodies against TIMP2 (Abcam, IgG2a, Mouse monoclonal, 1:250) and MMP2 (Abcam, IgG, Rabbit monoclonal, 1:250) were applied to slides, followed by goat anti-mouse IgG Alexa Fluor 596 (Abcam, IgG, Goat polyclonal, 1:400) or goat anti-rabbit IgG Alexa Fluor 647(Abcam, IgG, Goat polyclonal, 1:400). Finally, the sections were stained with Hoechst (11,000, Abcam) prior to be imaged by fluorescent microscope (Olympus). Then, the positive cell ratios of TIMP2 and MMP2 to evaluate expression levels were obtained by dividing the number of TIMP2- and MMP2-positive cells by the total number of cells in the defective area, respectively. The quantitative results were analyzed using ImageJ software with 3 randomly selected fields of each section and 3 sections per specimen.
4
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.
5
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.
6
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).
7
RBD-Specific IgG Antibody Quantification
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To evaluate RBD-specific IgG responses from mice sera, 100 ng/well of RBD-histag (GenScript, Singapore) in 50 mM bicarbonate buffer pH 9.6 were coated onto Nunc-Immuno MaxiSorp plates (Thermo Fisher Scientific, USA) at 4°C overnight. Then, the wells were blocked with blocking buffer and incubated at 37°C for 1 h followed by washing with PBST three times. 2-fold serially diluted mice sera in PBS (starting form 1:100) were added to the wells and incubated at 37°C for 1 h. After washing, the wells were incubated with 1:1000 diluted HRP-conjugated secondary antibodies including goat anti-mouse IgG(H+L) (Invitrogen, USA), IgG1 (Abcam, UK), or IgG2a (Abcam, UK) at 37°C for 1 h. Unbound antibodies were removed, and the reaction was developed using TMB chromogen solution (Invitrogen, USA) at room temperature for 20 min and then stopped by an equivalent volume of 1 N H2SO4. The absorbance at 450 nm were measured using a microplate reader CALIOstar (BMG Labtech, Germany). Anti-RBD IgG end-point titers were reported as a reciprocal of a highest dilution factor whose OD450 still exceeds a cut-off value. In this study, the cut-off value is defined as an average of OD450 from a negative control (blank using PBS instead of mice serum) + 8.042 x standard deviation [35 (link)].
8
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.
9
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).
10
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).
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