CD49b+ NK cells were enriched by magnetic sorting (Miltenyi) from BM. For stimulation through NK1.1, 10 µg/ml anti-NK1.1 (PK136, 108701; BioLegend) in NaHCO3 (pH 9.2) was precoated on an ELISA plate (655081; Greiner Bio-One). After culture in 20 ng/ml murine IL-15 (210-15; PeproTech) overnight, NK cells were then added to the plate with Brefeldin A (555029; BD Biosciences) for the last 4.5 h. For stimulation through cytokines, NK cells were cultured with 1,000 U/ml IL-2 (212-12; PeproTech) and 10 ng/ml IL-12 (210-12, PeproTech) overnight and Brefeldin A for the last 4.5 h. Cytofix/Cytoperm Fixation/Permeabilization Kit (554714; BD Biosciences) was used to detect IFN-γ (XMG1.2, 12-7311-81; eBioscience), and Cyto-Fast Fix/Perm Buffer Set (426803; BioLegend) was used to detect GZMB (QA16A02, 372207; BioLegend) according to the manufacturer’s instructions.
Elisa plate
Manufactured by Greiner
Sourced in Germany, Austria, United States, United Kingdom, Netherlands
ELISA plates are a type of laboratory equipment used for enzyme-linked immunosorbent assay (ELISA) testing. They are flat, multi-well plates made of plastic or glass, designed to hold small volumes of liquid samples for analysis. ELISA plates provide a standardized platform for conducting immunoassays, which are used to detect and measure the presence of specific proteins, antibodies, or other molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (3 mentions)
Fibrinogen, by Enzyme Research (3 mentions)
Fibronectin, by Enzyme Research (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Na2co3 solution, by Merck Group (2 mentions)
Polarstar omega, by BMG Labtech (2 mentions)
Synergy h4 plate reader, by Agilent Technologies (2 mentions)
Hrp coupled streptavidin, by BD (2 mentions)
Verikine mouse interferon alpha elisa kit, by PBL Assay Science (2 mentions)
Goat anti mouse igg h l secondary antibody conjugated to hrp, by Thermo Fisher Scientific (2 mentions)
Tmb substrate kit, by Thermo Fisher Scientific (2 mentions)
Sars cov 2 s1 protein, by ACROBiosystems (2 mentions)
Prism 4, by GraphPad (2 mentions)
Microsoft excel, by GraphPad (2 mentions)
Elx800 universal microplate reader, by Agilent Technologies (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Uv transparent plate, by Corning (2 mentions)
Multiscan go reader, by Thermo Fisher Scientific (2 mentions)
Collagen 1, by Merck Group (2 mentions)
Collagen 4, by Merck Group (2 mentions)
73 protocols using elisa plate
1
NK Cell Stimulation and Functional Assays
2
IL-17A Binding Affinity Assay
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50 µl of 5 nM hIL-17A was coated on the ELISA plate (Greiner Bio-One, Germany) for 1 h. Afterwards, the plate was washed 3× with 300 µl PBS-T/well, and the wells were blocked with PBS 1% BSA, and then incubated with 50ul of indicated antibody at a concentration range of 0 to 200 nM for 1 h. Subsequently, 50 µl of 25 nM of IL-17RA was added for 15 min and washed again three times. Secondary anti His HRP conjugated antibody (Santa Cruz Biotechnology, USA, cat: sc8036 HRP) diluted 1:250 in PBS-T was added for an incubation of 20 min at RT. Finally, the plate was washed three times with 300 μl PBS-T. The development using TMB and plate detection was performed as described for the polyspecificity assay.
3
SARS-CoV-2 Antibody ELISA Profiling
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All SARS-CoV2 recombinant proteins were purchased from Sino Biological (Beijing, China). For ELISA, 200 ng/well of WT SARS-CoV2 spike (40589-V08B1), spike S1 subunit (40591-V08H), spike S2 subunit (40590-V08B), NTD (40591-V49H), RBD (40592-V08H), nucleocapsid (40588-V08B) and envelope (40609-V10E3), Omicron spike (40589-V08H26), spike S1 subunit (40591-V08H41), RBD (40592-V08H121) and nucleocapsid (40588-V07E34) were coated on the 96-well ELISA plate (655061, Greiner Bio-one) using coating buffer (G3022, Saint Bio) overnight at 4 °C, respectively. Plates were washed by PBS supplemented with 0.5% Tween-20 (PBST) for three times, followed by blocking with 5% BSA in PBST (blocking buffer) for 1 h at room temperature. Sera were firstly diluted 40-fold, followed by 4-fold serial dilution and incubation at 4 °C overnight. Plates were washed 5 times by PBST, and incubated with 100 μl/well goat HRP conjugated anti-human IgG antibody (2040-05, SouthernBiotech, 1:3000) in PBST at room temperature for 30 min. After washing 5 times with PBST, 100 μl/well 3,3′,5,5′-Tetramethylbenzidine substrate (P0209, Beyotime) was added to each well for 15 min, stopped by the stopping buffer (P0215, Beyotime). OD450 was measured by Varioskan Lux Microplate Reader (Thermo Fisher).
4
PLGA-Based Drug Delivery System
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Poly (lactic-co-glycolic acid) (PLGA) (50:50, Mw = 12,000) was purchased from Boehringer Ingelheim (Ingelheim, Germany). Ranibizumab was a generous gift from Genentech (San Francisco, CA). Dichloromethane (DCM), acetonitrile, ethylene glycol (EG), and Dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific (Newton, NJ). The fluorescence dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (‘DiI’, D-282) was purchased from Invitrogen (Grand Island, NY) and Nile red from Sigma-Aldrich (St. Louis, MO). Rhodamine-6G was purchased from Acros organics (Fisher Scientific Newton, NJ). The Enzyme-linked immunosorbent assay (Elisa) plate was purchased from Greiner Bio-one (Monroe, NC).
5
Platelet-poor Plasma Hemoglobin Quantification
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1 mL aliquots of sheared blood samples were centrifuged at 4700 g for 7 min to prepare platelet-poor plasma (PPP). 100 µL PPP was transferred from each aliquot into a deep well 96-well plate (StarLabs, Milton Keynes, UK) and diluted with 1 mL 0.1% Na 2 CO 3 solution (Sigma-Aldrich). 170 µL diluted PPP was transferred into a 96-well flat bottom plate (ELISA plate, Greiner Bio-One, Stonehouse, UK) and the absorbance was measured at three wavelengths: 380, 415 and 450 nm (POLARstar Omega, BMG LABTECH Ltd, Aylesbury, UK). The plasma free haemoglobin (pfHb) was calculated (equation (1)) as described [31] .
6
Plasma-free Hemoglobin Quantification
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One ml blood samples were collected hourly and centrifuged at 4700 x g for 7 min. The plasma supernatant (100 µl) was transferred from each aliquot into a deep well 96-well plate (StarLabs, Milton Keynes, UK) and diluted with 1 ml 0.1% Na 2 CO 3 solution (Sigma-Aldrich, Poole, UK). 170 µl diluted plasma was transferred into a 96-well flat bottom plate (ELISA plate, Greiner Bio-One, Stonehouse, UK) and the absorbance was measured at three wavelengths: 380, 415, and 450 nm (POLARstar Omega, BMG LABTECH Ltd., Aylesbury, UK). The plasma-free haemoglobin (pfHb) was calculated by the Harboe direct spectrophotometric method (Eq. ( 1)) as described [37] . The normalized index of haemolysis (NIH) was calculated as described by (2) [36] .
7
Quantifying ECM Protein-Antibody Interactions
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Ninety-six-well ELISA plates (Greiner Bio-One) were coated with recombinant human ECM proteins (10 mg/ml), fibronectin (Sigma-Aldrich), fibrinogen (von Willebrand factor-and fibronectin-depleted; Enzyme Research Laboratories), osteopontin (Sigma-Aldrich), vitronectin (Sigma-Aldrich), collagen I (EMD Millipore), collagen II (EMD Millipore), collagen III (EMD Millipore), or collagen IV (EMD Millipore) in PBS for 1 hour at 37°C, followed by blocking with 2% BSA in PBS with 0.05% Tween 20 (PBS-T) for 1 hour at room temperature. Then, wells were washed with PBS-T and further incubated with PlGF-2 123-144 -Abs or unmodified Abs (10 mg/ml each) for 1 hour at room temperature. After three washes with PBS-T, wells were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated Abs against rat IgG or hamster IgG (Jackson ImmunoResearch). After washes, bound Abs were detected with tetramethylbenzidine sub-strate by measurement of the absorbance at 450 nm with subtraction of the absorbance at 570 nm.
8
Diverse T Cell Activation Protocols
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For non-specific TCR triggering, PBMCs, enriched CD8 T cells, sorted cells or colonic lymphocytes were stimulated with plate bound anti-CD3/28 antibodies (Miltenyi), or anti-CD3/28 beads (Miltenyi) at 1:1 ratio. ELISA plates (Greiner) were coated with 5 μg/mL anti-CD3/28 with the final volume of 100 μL at 4°C for overnight. Antibody mix was washed off the next morning, and plates were used after 1-hour 37°C incubation with R10. anti-CD3/28 beads were prepared following the manufacturer’s instructions.
MAIT-specific TCR triggering was achieved by co-culturing of 2x105 MACS-enriched CD8s with 1x105 THP1 cells which had been previously pulsed with 10nM 5-OP-RU (kindly provided by David Fairlie) for 2 hours. Unpulsed THP1s were used as controls.
For cytokine triggering, cells were stimulated for 20 hours with IL-12 (Miltenyi) at 2ng/mL, IL-18 (MBL) at 50ng/ml, IL-15 (Miltenyi) at 25ng/ml, TL1A (R&D) at 100ng/ml, unless otherwise stated.
For activation of MAIT cells by bacteria-derived ligands, THP1 cells were loaded with PFA-fixed (2%, 20min) E. coli (DH5α, Invitrogen) at a 25 bacteria per cell (BpC) ratio overnight. Bacterially loaded THP1s were washed and co-cultured with MACS-enriched CD8s at a 1:2 ratio. In order to block the TCR-dependent component of this activation, in some experiments an anti-MR1 blocking antibody (26.5, Biolegend) was added to the co-cultures.
MAIT-specific TCR triggering was achieved by co-culturing of 2x105 MACS-enriched CD8s with 1x105 THP1 cells which had been previously pulsed with 10nM 5-OP-RU (kindly provided by David Fairlie) for 2 hours. Unpulsed THP1s were used as controls.
For cytokine triggering, cells were stimulated for 20 hours with IL-12 (Miltenyi) at 2ng/mL, IL-18 (MBL) at 50ng/ml, IL-15 (Miltenyi) at 25ng/ml, TL1A (R&D) at 100ng/ml, unless otherwise stated.
For activation of MAIT cells by bacteria-derived ligands, THP1 cells were loaded with PFA-fixed (2%, 20min) E. coli (DH5α, Invitrogen) at a 25 bacteria per cell (BpC) ratio overnight. Bacterially loaded THP1s were washed and co-cultured with MACS-enriched CD8s at a 1:2 ratio. In order to block the TCR-dependent component of this activation, in some experiments an anti-MR1 blocking antibody (26.5, Biolegend) was added to the co-cultures.
9
Glycopeptide Library Binding Analysis
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The library was screened
against biotin-SBA and biotin-VVA using an enzyme-linked lectin assay
(ELLA), as described previously.52 (link),53 (link) A high-binding
96-well enzyme-linked immunosorbent assay (ELISA) plates (Greiner
Bio-One) was coated with the glycopeptide sublibraries and controls
(50 μL per well) at four different concentrations (0.1, 0.5,
10, and 500 μg/mL) in phosphate-buffered saline (PBS) (0.01
M, pH 7.4). Wells were incubated overnight to dry at 37 °C, and
then blocked with 3% bovine serum albumin (BSA) in PBS (300 μL)
overnight on the shaker. The coated wells were incubated with either
biotin-conjugated SBA or VVA lectin (100 μL, 50 μg/mL
in PBS) for 2 h at room temperature on the shaker. Upon washing with
PBS (2×, 100 μL), wells were treated with horseradish peroxidase
(HRP) conjugated streptavidin (100 μL, 1:4000) for 1 h at room
temperature on the shaker. The wells were then washed again with PBS
(2×, 100 μL) and water (300 μL), upon which 3,3,5,5-tetramethylbenzidine
(TMB) was added (100 μL) and incubated at room temperature for
15 min on the shaker. The reaction was terminated using 2 M sulfuric
acid (100 μL). The absorbance readings were recorded at 450
nm using an ELISA plate reader (BioTek, EPOCH plate reader). The average
absorbance reading after background subtraction was plotted against
the glycopeptide library concentration.
against biotin-SBA and biotin-VVA using an enzyme-linked lectin assay
(ELLA), as described previously.52 (link),53 (link) A high-binding
96-well enzyme-linked immunosorbent assay (ELISA) plates (Greiner
Bio-One) was coated with the glycopeptide sublibraries and controls
(50 μL per well) at four different concentrations (0.1, 0.5,
10, and 500 μg/mL) in phosphate-buffered saline (PBS) (0.01
M, pH 7.4). Wells were incubated overnight to dry at 37 °C, and
then blocked with 3% bovine serum albumin (BSA) in PBS (300 μL)
overnight on the shaker. The coated wells were incubated with either
biotin-conjugated SBA or VVA lectin (100 μL, 50 μg/mL
in PBS) for 2 h at room temperature on the shaker. Upon washing with
PBS (2×, 100 μL), wells were treated with horseradish peroxidase
(HRP) conjugated streptavidin (100 μL, 1:4000) for 1 h at room
temperature on the shaker. The wells were then washed again with PBS
(2×, 100 μL) and water (300 μL), upon which 3,3,5,5-tetramethylbenzidine
(TMB) was added (100 μL) and incubated at room temperature for
15 min on the shaker. The reaction was terminated using 2 M sulfuric
acid (100 μL). The absorbance readings were recorded at 450
nm using an ELISA plate reader (BioTek, EPOCH plate reader). The average
absorbance reading after background subtraction was plotted against
the glycopeptide library concentration.
10
Quantitative ELISA for IgG Antibodies
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IgG ELISA was performed by coating ELISA plates (Greiner Bio‐One, Alphen a/d Rijn, The Netherlands) with 0.3 µg/well of A/PR/8, H1N1pdm or X‐31 WIV in coating buffer (17.8 mM Na2CO3, 22.5 mM NaHCO3, pH9.6) overnight at 37°C. ELISA was done as described previously.23 IgG titers were calculated as log10 of the reciprocal of the sample dilution corresponding to an absorbance of 0.2 at the wavelength of 492 nm. LoD was calculated using the first dilution made and the negative samples were given half the value of the LoD value.
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