Near-saturation Akata and Mutu I cell cultures were diluted with equal volumes of fresh RPMI 1640 (with 10% FBS and 0.5% pen/strep) one day before induction. To induce reactivation, cells were pelleted and resuspended at a concentration of 106 cells/ml in fresh RPMI 1640 (with 10% FBS and 0.5% pen/strep) plus either 10μg/ml anti-human IgG (Sigma-Aldrich, catalog no. I2136) (for Akata cells) or 10 μg/ml anti-human IgM (Sigma-Aldrich, catalog no. I0759) (for Mutu cells). For Pacific Biosciences Iso-Seq, cells were harvested at 20 and 24 h post-induction, for Illumina RNA-Seq, cells were harvested at 0 min, 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h post-induction and for deepCAGE, cells were harvested at 24 h post-induction. For qRT-PCR, cells were harvested at 0 and 24 h (Akata) or 0 and 48 h (Mutu).
Anti human igm
Manufactured by Merck Group
Sourced in United States
Anti-human IgM is a laboratory reagent used to detect and measure the presence of immunoglobulin M (IgM) antibodies in biological samples. IgM antibodies are the first type of antibodies produced by the body in response to an infection or antigen exposure. The anti-human IgM reagent can be used in various immunoassay techniques to identify and quantify IgM levels, which can provide information about the stage of an immune response or the presence of certain diseases.
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Lab products found in correlation
Anti human igg hrp, by Thermo Fisher Scientific (3 mentions)
Spectramax microplate reader, by Molecular Devices (3 mentions)
Multiskan sky, by Thermo Fisher Scientific (3 mentions)
Iso seq, by Illumina (1 mentions)
Rna seq, by Illumina (1 mentions)
Tmb substrate solution, by Merck Group (1 mentions)
Microfluor plates, by Thermo Fisher Scientific (1 mentions)
Victor 3v, by PerkinElmer (1 mentions)
Anti actin a2066, by Merck Group (1 mentions)
Anti cdc25a f 6, by Santa Cruz Biotechnology (1 mentions)
Anti chk1 g 4, by Santa Cruz Biotechnology (1 mentions)
Infinite m200 plate reader, by Tecan (1 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Hrp conjugated monoclonal anti his tag antibodies, by Merck Group (1 mentions)
Hrp conjugated anti hamster igg, by Merck Group (1 mentions)
Hrp conjugated anti human igg, by Merck Group (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Fish gelatin, by Merck Group (1 mentions)
Alkaline phosphatase conjugated anti human igm, by Merck Group (1 mentions)
Purified human igm, by Merck Group (1 mentions)
15 protocols using anti human igm
1
Epstein-Barr Virus Reactivation Assay
2
Rapid LPS Detection Lateral Flow Assay
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Fifty micrograms of extracted LPS were placed at equal distance as an array on the test (T) area and 2 μg of anti-human IgM (Sigma Aldrich, USA) was placed as an internal control (IC). The membrane was dried in a desiccator for 3 hours at 37°C. The unconjugated regions were blocked in 10 mM phosphate buffer (pH 7.2) containing 1% non-fat milk for 1 hour, washed twice with the same buffer and dried at room temperature. Glass fiber conjugate pads (GE healthcare) were dipped in gold-conjugated protein A dissolved in 2mM borate buffer (pH 7.2) supplemented with 5% sucrose. The pads were then air dried at 37°C for 2 h. Cellulose fiber sample pads (BioRad, Hercules, CA, USA) were dipped in sample pad buffer (50 mM borate buffer (pH 7.2), 5% sucrose, 0.5% Tween 20, 5% dextran, and 0.1% skim milk) and dried at 50°C [19 (link)]. Fifty microliters of patient sera was placed on the sample pad and incubated for 10 minutes. Positivity was determined by the development of two spots in the internal control (IC) and test (T) area. Negative samples developed a single spot in IC area alone.
3
SARS-CoV-2 Spike Protein ELISA
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Example 8
For SARS CoV or SARS-CoV-2 spike protein binding ELISAs, 96-well plates (Corning; Cat #3690) were coated with 5 μg/ml of SARS CoV or SARS-CoV-2 spike protein diluted in PBS and incubated overnight at 4° C. Wells were washed and then blocked with 5% non-fat dried milk (NFDM) in PBS for 1 hour at 37° C. Wells were washed 3 times with PBS and serial dilutions of human plasm in 5% NFDM-PBS were added and incubated for 1 hour at 37° C. Plates were then washed 3 times with PBS and secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher Scientific; cat #31413) or anti-human-IgM (Sigma Aldrich; cat #AP114P) detection antibodies were added at 1:8000 dilution in 5% NFDM-PBS for 1 hour at 37° C. After washing 3 times with PBS detection reagent was added per manufacturer recommendations (Thermo Scientific; Cat #34029) and absorbance was measures at 450 nM wavelength using a Spectramax microplate Reader (Molecular Devices).
4
SARS-CoV-2 Spike Protein ELISA
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Example 8
For SARS CoV or SARS-CoV-2 spike protein binding ELISAs, 96-well plates (Corning; Cat #3690) were coated with 5 μg/ml of SARS CoV or SARS-CoV-2 spike protein diluted in PBS and incubated overnight at 4° C. Wells were washed and then blocked with 5% non-fat dried milk (NFDM) in PBS for 1 hour at 37° C. Wells were washed 3 times with PBS and serial dilutions of human plasm in 5% NFDM-PBS were added and incubated for 1 hour at 37° C. Plates were then washed 3 times with PBS and secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher Scientific; cat #31413) or anti-human-IgM (Sigma Aldrich; cat #AP114P) detection antibodies were added at 1:8000 dilution in 5% NFDM-PBS for 1 hour at 37° C. After washing 3 times with PBS detection reagent was added per manufacturer recommendations (Thermo Scientific; Cat #34029) and absorbance was measures at 450 nM wavelength using a Spectramax microplate Reader (Molecular Devices).
5
SARS-CoV-2 Spike Protein ELISA
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Example 8
For SARS CoV or SARS-CoV-2 spike protein binding ELISAs, 96-well plates (Corning; Cat #3690) were coated with 5 μg/ml of SARS CoV or SARS-CoV-2 spike protein diluted in PBS and incubated overnight at 4° C. Wells were washed and then blocked with 5% non-fat dried milk (NFDM) in PBS for 1 hour at 37° C. Wells were washed 3 times with PBS and serial dilutions of human plasm in 5% NFDM-PBS were added and incubated for 1 hour at 37° C. Plates were then washed 3 times with PBS and secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher Scientific; cat #31413) or anti-human-IgM (Sigma Aldrich; cat #AP114P) detection antibodies were added at 1:8000 dilution in 5% NFDM-PBS for 1 hour at 37° C. After washing 3 times with PBS detection reagent was added per manufacturer recommendations (Thermo Scientific; Cat #34029) and absorbance was measures at 450 nM wavelength using a Spectramax microplate Reader (Molecular Devices).
6
SARS-CoV-2 RBD Antibody ELISA Assay
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All ELISA assays were performed on a DSX automated ELISA system device (DYNEX Technologies). The in‐house assay was prepared as follows: 96‐well plates were coated overnight at 4°C with 100 µL of 1 µg/mL RBD protein in PBS. The following day, each well was blocked with 300 µL of PBS/0.15% casein at 4°C until use and at least overnight. Subsequently, plates were washed twice with PBS and 100µl sera were added at a 1:100 dilution in PBS/0.15% casein for 1 hour at RT. After five washes with 300 µL PBS/0.1% Tween, 100 µL of HRP‐labeled secondary polyclonal anti‐human IgM (Sigma, A0420) and anti‐human IgG (Sigma, A0170) antibodies was added in a 1:10’000 dilution for 30 minutes at RT. Again, the plates were washed 5 times with PBS/0.1% Tween and 100 µL of TMB substrate solution (Sigma, T4444) was added for 15 minutes at RT. The development was stopped by adding 100 µL of 0.5M H2SO4, and results were measured at OD450‐620nm. All samples with an OD > 0.5 were assigned as positive.
7
Quantifying Plasma Antibodies by Chemiluminescence ELISA
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Plasma antibody levels from human and mouse were measured with a chemiluminescence immunoassay (enzyme-linked immunosorbent assay, ELISA). Antigen was dissolved in PBS (5 μg/ml) and incubated at +4°C overnight in MicroFluor plates (Thermo Scientific, Rockford, IL, USA). Automated plate washer was used to wash the plates three times with PBS containing 0.27mM ethylenediaminetetraacetic acid (EDTA). Plates were blocked with PBS-EDTA containing 0.5% fish gelatin (Fg) and incubated for one hour at room temperature. Plasma samples were diluted with 0.5% Fg-PBS-EDTA and the antibody levels were measured with alkaline phosphatase-labeled antibodies (anti-human-IgG, anti-human-IgM, anti-mouse-IgG, anti-mouse-IgM) diluted according to manufacturer specifications (Sigma-Aldrich). Chemiluminescence was detected using LumiPhos 530 (33% Lumigen) substrate, measured with luminescence multilabel counter (PerkinElmer Victor3V), and expressed in relative light units (RLU).
Specific binding of plasma antibodies to Aa-HSP60 and MAA-LDL was tested with competitive chemiluminescence immunoassay. Aa-HSP60 and MAA-LDL (0–100 μg/mL) were incubated with plasma samples overnight at +4°C. Incubated solutions were centrifuged at 16,000 × g at +4°C and the remaining antibody levels measured with ELISA as described above.
Specific binding of plasma antibodies to Aa-HSP60 and MAA-LDL was tested with competitive chemiluminescence immunoassay. Aa-HSP60 and MAA-LDL (0–100 μg/mL) were incubated with plasma samples overnight at +4°C. Incubated solutions were centrifuged at 16,000 × g at +4°C and the remaining antibody levels measured with ELISA as described above.
8
Antibody and Inhibitor Assay Protocol
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The following antibodies and inhibitors were used: anti-AID (EK2 5G9), anti-CDC25A (F-6, Santa Cruz), anti-Chk1 (G-4, Santa Cruz), anti-Actin (A2066, Sigma-Aldrich), anti-human IgM (P9295, Sigma-Aldrich), UCN-01 (U6508, Sigma-Aldrich) and TCS2312 (TOC-3038, Tocris).
9
SARS-CoV-2 Antibody Detection by ELISA
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Enzyme-linked immunosorbent assay (ELISA) was performed using 96-well high- binding half-area polystyrene plates coated overnight at 4°C with 4 μg/ml of Spike protein, 2 μg/ml Nucleocapsid protein (NP) (Kindly provided by Dr. Ricardo Gazzinelli, UFMG, Brazil) or 0.8 μg/ml of the RBD domain from SARS-CoV-2 were all expressed in HEK293T cells (12 (link)). In short, 50 µl of diluted sera (1:100) were incubated at 37°C for 45 min. Peroxidase-conjugated goat anti-human IgG (BD Pharmingen,USA), anti-human IgA (KPL, USA) or anti-human IgM (Sigma, USA) secondary antibody conjugates were diluted 1:10,000, and incubated at 37°C for 30 min. The optical density (OD) at 492 nm was measured with a microplate reader (Epoch, BioTek, USA). Values were determined as OD minus blank and cutoff was determined as the average OD of 12 samples pre-pandemics + 3× standard deviation. Results are given as the ratio of OD sample/cutoff. An antibody ratio of ≥1.2 was considered positive.
10
Stimulating B-cell Responses
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Akata and Mutu I cells were spun down and resuspended at a concentration of 106 cells/ml in fresh RPMI 1640 medium (10%FBS). Anti-human IgG (Sigma-Aldrich, catalog no. I5260) or anti-human IgM (Sigma-Aldrich, catalog no. I0759) was added to Akata and Mutu I cell suspensions, respectively, to a final concentration of 10 ug/ml. Treated and untreated cells were harvested 24 h and 48 h later for Akata and Mutu I cells, respectively, and subjected to RNA isolation.
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