Intracellular cytokine staining was performed using 4‐week expanded peptide‐specific T cells. Cell mixtures, each consisting of 1 × 106 expanded peptide‐specific cells and 1 × 106 autologous PBMCs, were cultured with 5 µg/mL of the respective peptide or medium only in a 48‐well plate overnight at 37℃ with CO2. After peptide stimulation, 5 µg/mL of brefeldin A (Bio‐Rad) was added to the cells, followed by incubation for 4 hours at 37℃. Stimulated cells were collected and stained with 5 µg/mL of BoLA class II tetramer–PE in cRPMI for 1 hour at room temperature and then with anti‐bovine CD4‐Alexa 647 mAb in cRPMI for 20 min at room temperature. Stained cells were fixed and permeabilized using Leucoperm™ (Bio‐Rad) after washing twice with 2% FBS/PBS. Permeabilized cells were immediately stained with 1 µg/mL of mouse anti‐bovine IFN‐γ‐FITC (cc302, IgG1, Bio‐Rad), anti‐bovine IL‐4 FITC (CC303, IgG2a, Bio‐Rad), anti‐human IL‐17A FITC (MT504, IgG1, Mabtech) or mouse isotype control antibody‐FITC (IgG1 and IgG2a, Bio‐Rad) in cRPMI for 20 min at room temperature. After washing cells twice, cells were suspended in 1% PFA/PBS and immediately analysed by flow cytometry.
Igg2a
Manufactured by Bio-Rad
Sourced in United Kingdom
IgG2a is a specific type of immunoglobulin (antibody) molecule produced by the immune system. It is a class of IgG antibodies, which are the most abundant antibody type in the body. IgG2a antibodies play a role in the immune response, but their core function is not interpreted in this description.
Automatically generated - may contain errors
Lab products found in correlation
Igg2b, by Bio-Rad (2 mentions)
Biotin labeled rat anti mouse igg1, by Bio-Rad (2 mentions)
Leucoperm, by Bio-Rad (1 mentions)
Streptavidin coated 96 well plate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated rabbit anti mouse igg, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Biotinylated anti mouse igg1, by Bio-Rad (1 mentions)
Igg2a fitc, by Bio-Rad (1 mentions)
Donkey anti rabbit igg, by GE Healthcare (1 mentions)
Hrp labeled goat anti mouse igg, by GE Healthcare (1 mentions)
Streptavidin hrp, by Bio-Rad (1 mentions)
Carbonate bicarbonate buffer ph 9, by Merck Group (1 mentions)
Maxisorp immunoplate, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg1, by Bio-Rad (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Anti mouse f4 80, by Thermo Fisher Scientific (1 mentions)
Bz 9000 fluorescent microscope, by Keyence (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facsaria, by BD (1 mentions)
13 protocols using igg2a
1
Intracellular Cytokine Profiling of Peptide-Specific T Cells
2
Quantifying Antigen-Specific Antibody Responses
3
Quantifying Antibody Responses to Immunization
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Serum total IgG titers,
isotype IgG1 and IgG2A titers, and avidity indices
were determined as previously described.24 Briefly, 96-well plates were coated with OVA, blocked with bovine
serum albumin, incubated with serially diluted serum, and detected
with HRP-labeled antimouse IgG, IgG1, or IgG2A (Bio-Rad) followed by development and measurement of optical absorbance
at 450 nm. Antibody titer is reported as reciprocal serum dilution
at an absorbance of 0.5. For avidity indices, duplicate serum dilutions
were prepared for each sample, and for one set of dilutions, wells
were incubated for 10 min with 6 M urea prior to detection with the
respective anti-mouse secondary antibody. The avidity index is reported
as the ratio of the titers of the urea-treated sample to those of
the non-urea-treated sample.
isotype IgG1 and IgG2A titers, and avidity indices
were determined as previously described.24 Briefly, 96-well plates were coated with OVA, blocked with bovine
serum albumin, incubated with serially diluted serum, and detected
with HRP-labeled antimouse IgG, IgG1, or IgG2A (Bio-Rad) followed by development and measurement of optical absorbance
at 450 nm. Antibody titer is reported as reciprocal serum dilution
at an absorbance of 0.5. For avidity indices, duplicate serum dilutions
were prepared for each sample, and for one set of dilutions, wells
were incubated for 10 min with 6 M urea prior to detection with the
respective anti-mouse secondary antibody. The avidity index is reported
as the ratio of the titers of the urea-treated sample to those of
the non-urea-treated sample.
4
Quantifying Thawed LNP-RNA Vaccine Efficacy
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To quantitatively assess the efficacy of thawed LNPs compared with freshly prepared samples using repRNA encoding for the HIV env trimer, we vaccinated healthy Balb/C mice by injecting 1 μg RNA doses of the LNP-RNA i.m. in each of the left and right gastrocnemius muscle. At weeks 2 and 4 post-i.m. injection, the mice underwent retro-orbital bleeding; blood was collected in Z-gel PP tubes for blood serum collection (CAT#41.1500.005; Sarstedt). Serum was collected by centrifuging blood at 10,000 ×g for 4 min, and stored at −80 °C prior to use. To conduct ELISAs, NUNC MaxiSorp plates were coated overnight with 1 μg/mL purified HIV antigen in PBS, then blocked for 2 h with 10% BSA in PBS. Mouse sera were initially diluted 50× in blocking buffer, followed by 3× serial dilutions. Diluted sera were transferred to blocked plates and incubated for 2 h. HRP-conjugated immunoglobulins (e.g., IgG, IgG1, IgG2a, IgG2b, IgG3, IgM; Bio-Rad) were used as detection antibodies at 1:5000 for endpoint titer assessments, with gp120-specific monoclonal antibody VRC01 used as a positive control. 3,3′,5,5’-Tetramethylbenzidine (TMB) signal was read using a microplate reader by subtracting the absorbance at 450 nm by that at 550 nm.
5
Humoral Immune Response Analysis
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Humoral immune responses elicited by ProR peptide/rCsHSPs complexes were analyzed by detecting the production of specific antibodies against the peptide in CsProR peptide-coated ELISA plates. Briefly, streptavidin-coated 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) were incubated with 5 μg/ml biotin-conjugated CsProR peptide for 18 h at 4 °C. All subsequent steps were carried out at room temperature. After three washes with PBS containing 0.1% Triton X-100 (PBST), plates were blocked with blocking buffer (5% skim milk in PBST) for 2 h, washed, and incubated in immunized mouse serum diluted in blocking buffer for 2 h. After washing, 100 μl/well horseradish peroxidase-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) diluted 1:10,000 in blocking buffer were added, followed by incubation for 2 h; 50 μl/well 3,3′,5,5′-tetramethylbenzidine/H2O2 substrate were added, followed by incubation for 30 min. The enzymatic reaction was terminated by adding 50 μl/well of 2 N H2SO4 to the wells and optical density was measured with a spectrophotometer at 450 nm (reference wavelength: 620 nm). For measuring IgG isotypes, detection of antibodies was determined using either horseradish peroxidase-conjugated goat anti-mouse IgG1 or IgG2a (BioRad, Hercules, CA, USA).
6
Quantifying LiChimera-specific Antibody Responses
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LiChimera-specific IgG, IgG1, and IgG2a production was determined through ELISA in sera collected from all experimental mice groups at predetermined time points. Sera samples were added at a dilution of 1:400 for 90 min in 96-well microtiter plates pre-coated with 2 μg/mL of LiChimera. After that, HRP-conjugated anti-mouse IgG (1:5000 dilution) (Thermo Scientific) or biotinylated anti-mouse IgG1 (1 μg/mL) and IgG2a (250 ng/mL) (both obtained from AbD Serotec, Oxford, UK) were added for 1 h, at 37 °C. In the case of biotinylated antibodies streptavidin-HRP at a dilution of 1:5000 was added and samples were incubated for another 1 h, at 37 °C. The enzyme-labeled complexes were detected by reaction with TMB substrate. The absorbance was measured at 450 nm using an ELISA microplate spectrophotometer (MRX). In some cases, detection of the parasite-specific IgG1 and IgG2a antibodies were also conducted according to a previously published protocol for mice [39 (link)].
7
T Cell Activation and Signaling Assay
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CD3 MEM-57 (Abcam), CD59 MEM-43, and CD71 monoclonal antibodies (mAb), or appropriate isotype control IgG2a (all AbD Serotec) were used for cell stimulation. For Western blotting β-actin (C4) mAb, CD3-zeta mAb (F-3), LAT mAb (4i355), Lck mAb (3A5) (all SantaCruz Biotechnology), CD3ε pAb (Sigma-Aldrich), CD59 pAb (both Abcam), Fyn mAb (FYN-01) (Exbio Praha), and HRP-labeled goat anti-mouse (Sigma-Aldrich) or donkey anti-rabbit IgG (GE Healthcare) were used. Fluorophore-conjugated antibodies CD3-FITC MEM-57, CD59-FITC MEM-43, and IgG2a-FITC were purchased from AbD Serotec, CD8a-FITC OKT-8 from eBioscience®, CD3-AF-647 MEM-57, and IgG2a-AF647 were from Exbio Praha. For inhibition of Src family kinases, cells were incubated with 10 µM PP2 (Molecular Probes) for 30 min at room temperature.
8
Quantification of Peptide-Specific Antibodies
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Blood collected from each group of mice (n = 8/group) at fifteenth day post the third immunization, were centrifuged at 4000 × g for 5 min and separated sera were aliquoted for the detection of specific antibodies against each synthetic multi-epitope peptide by specific ELISAs as previously described (50 (link)). In brief, 96-well microtiter plates were coated with 5 μg/ml of each individual peptide in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3), pH 9.6 and left overnight at 4°C. For the detection of total IgG antibodies, 10-fold dilutions of each serum sample in 1% BSA in PBS-T were added and incubated with HRP-labeled goat anti-mouse IgG (1/1000 dilution; GE Healthcare, Buckinghamshire, UK). For the detection of IgG1 and IgG2a isotypes, serum samples (1/100 dilution) were added and plates were similarly incubated either with biotin-labeled rat anti-mouse IgG1 (500 ng/ml; AbD Serotec, Oxford, UK) or IgG2a (250 ng/ml; AbD Serotec) followed by the addition of streptavidin–HRP (1/5000 dilution; AbD Serotec) and incubation for 1 h at 37°C. The cut-off value was determined as the mean OD value of normal mouse serum in a 1/100 dilution + 2SD.
9
ELISA for antibody detection
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MaxiSorp immunoplates (Nunc, Roskilde, Denmark) were coated with 100 μl/well 1 μg/ml of P0, P1, P2, P0-(P1-P2)2, or Msp-119 proteins dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6) (Sigma) overnight at 4°C. To titrate the sera, depending on the antibody class and subclass, 1 : 2, 1 : 3, or 1 : 4 serial dilutions in PBS-T were assayed (IgG and IgG1—1 : 64/1 : 104976; IgG2a—1 : 27/1 : 13122). Appropriate dilutions of secondary anti-mouse antibodies conjugated with alkaline phosphatase (100 μl/well) were added: IgG (1 : 3000), IgM (Sigma), IgG1, and IgG2a (1 : 2000) (AbD Serotec). The serum dilution corresponding to a mean absorbance value of 0.1 was considered the end-point titer.
10
IgG Subclass Profiling by ELISA
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The profiles of specific IgG subclasses in antisera were determined by ELISA. Briefly, microtitre plates were coated with Polypeptides or the individual immunodominant epitope 1 μg/well overnight at 4 °C. After blocking with 2% BSA (v/v), Polypeptides or the individual immunodominant epitope-specific antisera were added at a dilution of 1:500 (data at the factor of 1:250, 1:1000 and 1:2000 not shown) and incubated for 1 h at 37 °C. Normal mouse sera (pre-immune sera) served as a negative control. After washing, IgG isotype-specific primary antibodies (goat anti-mouse IgG1, IgG2a, IgG2b and IgG3, purchased from AbD Serotec) were added to the wells at 1:3,000 dilution and incubated for 1 h at 37 °C. After extensive washing, tetramethyl benzidine (TMB) substrate was added for 10 min at room temperature, and the reaction was stopped by addition of 100 μl 2 M sulfuric acid. Endpoint absorbances were read at 450 nm using a microplate reader (Bio-Rad).
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