Hybridoma cell supernatants were screened for PFD1235w DBLβ3_D4-reactive Abs using ELISA. Duplicate wells of MaxiSorp microtiter plates (Nunc) were coated with DBLβ3_D4 (50 μl; 1 μg/ml; 0.1 M glycine/HCl buffer pH 2.75; overnight; 4°C) and blocked with blocking buffer (PBS, 0.5 M NaCl, 1% Triton X-100, 1% BSA, pH 7.2). A total of 100 μl undiluted cell supernatant was added (1 h; room temperature). The plates were washed in PBS + 1% Triton X-100, and bound Ab was detected with an anti-mouse Ig-HRP (Dako; 1:3000 in blocking buffer). After 1 h of incubation, plates were developed using OPD tablets (Dako) according to the manufacturer’s instructions. The OD value was read at 490 nm using a VERSAmax microplate reader (Molecular Devices) and Softmax Pro v4.7.1.
Opd tablets
Manufactured by Agilent Technologies
Sourced in Germany, Denmark
OPD tablets are a laboratory product manufactured by Agilent Technologies. They are used as a reagent in various analytical procedures. The core function of OPD tablets is to provide a source of o-phenylenediamine, a chemical compound used in colorimetric detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated avidin, by Thermo Fisher Scientific (3 mentions)
Maxisorp microtiter plate, by Thermo Fisher Scientific (2 mentions)
Anti mouse ig hrp, by Agilent Technologies (2 mentions)
Mab1857, by R&D Systems (2 mentions)
Carbonate bicarbonate buffer, by Merck Group (2 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions)
Softmax pro v4, by Molecular Devices (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Cary 50 mpr microplate reader, by Agilent Technologies (1 mentions)
Endogen, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
10 protocols using opd tablets
1
Screening Hybridoma Supernatants for Anti-DBLβ3_D4 Antibodies
2
Serum Lcn-2 Quantification in CLP Mice
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Serum was collected from Sham-operated or CLP-treated mice at the indicated timepoints. A volume of 100 µL of each sample was applied to an ELISA well-plate previously covered with the anti-Lcn-2 (MAB1857, R & D) and blocked for 1 h. After sample incubation, the detection anti-Lcn-2 antibody was added. HRP-conjugated avidin (Invitrogen, Dreieich, Germany) was incubated for 1 h, the color reagent (OPD tablets; Dako, Jena, Germany) was added, and color development was assessed.
3
Screening for Anti-PFD1235w DBLβ3_D4 Antibodies
4
Quantifying Lcn-2 Secretion in MΦ
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Supernatants were collected from cultured MΦ. A volume of 100 µL of each sample was applied to an ELISA well-plate previously covered with the anti-Lcn-2 (MAB1857, R&D) and blocked for 1 h. After sample incubation, the detection anti-Lcn-2 antibody was added. HRP-conjugated avidin (Invitrogen, obtained from Thermo Fisher) was incubated for 1h, the color reagent (OPD tablets; Dako, obtained from Agilent, Waldbronn, Germany) was added, and the color development was assessed.
5
Apolipoprotein E Complement Binding Assay
6
Quantification of uPAR in Tumor Lysates
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Tumours were homogenized with the Precellys (Bertin technologies, Montigny le Bretonneux, France) and a suspensions containing the tumor lysate were stored at -80°C. The plate was coated with an anti uPAR antibody R2 (3μg/ml)[24 (link)] overnight at 4°C. After incubation, 2% BSA was added for 5 min and the plate was washed with buffer. uPAR standard (10 ng/ml) or tumor lysate (diluted 1:20) was added and incubated for 2 h in RT and washed with buffer. A primary antibody (rabbit-anti-uPAR, 1μg/ml) was added to the well and incubated for 30 min in RT and washed. A secondary HRP conjugated anti-rabbit antibody was added (diluted 1:2.500) and incubated for 30 min in RT and washed. The bound HRP conjugated antibody was quantified by adding the substrate (4 OPD tablets in 12 ml, Dako) and left to react until a proper level of color development was achieved. An ELISA reader was used to analyze the plate at 490 nm and 650 nm as reference.
7
Apolipoprotein E Complement Binding Assay
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Recombinant or plasma-purified ApoE (0.1, 0.4 or 0.5 μM), ApoE
peptides (0.1 μM), IgM (1 μg/ml), LDL, oxLDL, and MDA-LDL (each 1
mg/ml) or gelatin (10 μg/ml) were immobilized in carbonate-bicarbonate
buffer (Sigma) on microtiter plates (F96 Maxisorb, Nunc-Immuno module) overnight
at 4° C. After washing the plate three times with washing buffer (PBS)
containing 0.1% Tween 20) wells were blocked (PBS, 1% BSA, 5% milk) for at least
1 h at 37° C. Coated proteins were incubated for 1 h at 37° C with
C3, C3b, and C1q (0.1 μM) or C1q (0.02– 5.33 nM) or NHS (0.075
– 10%) in GVB++ buffer. Calcium-dependent binding of C1q to
ApoE was determined by diluting C1q in GVB++ buffer or in PBS and
adding increasing amounts of EGTA (3 – 12 μM) (Sigma) with a fixed
concentration of C1q (2 or 5 nM) to immobilized ApoE proteins. Binding force was
analyzed incubating C1q or the LDLR (0.1 μM) with or w/o NaCl (0.5 M) or
SDS (1 %) on immobilized ApoE3. C1q, C3, and C3b binding was analyzed by
specific primary antibodies and followed by HRP-conjugated secondary antibodies.
The reaction was developed with TMB (Kementec Diagnostics) or
1,2-phenylenediamine dihydrochloride (OPD tablets, Dako) and the absorbance at
450 nm or 492 nm was recorded.
peptides (0.1 μM), IgM (1 μg/ml), LDL, oxLDL, and MDA-LDL (each 1
mg/ml) or gelatin (10 μg/ml) were immobilized in carbonate-bicarbonate
buffer (Sigma) on microtiter plates (F96 Maxisorb, Nunc-Immuno module) overnight
at 4° C. After washing the plate three times with washing buffer (PBS)
containing 0.1% Tween 20) wells were blocked (PBS, 1% BSA, 5% milk) for at least
1 h at 37° C. Coated proteins were incubated for 1 h at 37° C with
C3, C3b, and C1q (0.1 μM) or C1q (0.02– 5.33 nM) or NHS (0.075
– 10%) in GVB++ buffer. Calcium-dependent binding of C1q to
ApoE was determined by diluting C1q in GVB++ buffer or in PBS and
adding increasing amounts of EGTA (3 – 12 μM) (Sigma) with a fixed
concentration of C1q (2 or 5 nM) to immobilized ApoE proteins. Binding force was
analyzed incubating C1q or the LDLR (0.1 μM) with or w/o NaCl (0.5 M) or
SDS (1 %) on immobilized ApoE3. C1q, C3, and C3b binding was analyzed by
specific primary antibodies and followed by HRP-conjugated secondary antibodies.
The reaction was developed with TMB (Kementec Diagnostics) or
1,2-phenylenediamine dihydrochloride (OPD tablets, Dako) and the absorbance at
450 nm or 492 nm was recorded.
8
Quantifying SALSA Levels in Biological Samples
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To quantify the levels of SALSA, samples were diluted and coated directly onto Maxisorp plates (Nunc, Denmark). SALSA purified from saliva was used as a protein concentration standard. After coating, the plates were blocked with 5% nonfat milk in TBS/1 mM Ca2+. The plates were washed with TBS/Ca2+ and 0.05% Tween-20 (TBS/Ca2+/Tween). SALSA levels were detected using monoclonal anti-SALSA (Hyb 213–06, Bioporto, Denmark) diluted to 0.05 μg/ml and HRP-conjugated rabbit anti-mouse antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:10 000 in TBS/Ca. OPD tablets (Dako, Denmark) were used for development and the color reaction was measured with an iEMS Reader MF (Labsystems, Espoo, Finland) at an OD of 492 nm. For each sample a dilution series was made to ensure that the readings were within the linear range. Measurements were based on a minimum of three separate assays.
9
Quantitative Complement C4d Assay
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Maxisorp 96-well plates (Nunc, Roskilde, Denmark) were coated overnight at room temperature with 3.5 g/ml of affinity-purified C4d neoepitope antibody diluted in PBS + 0.02% NaN3. Wells were blocked with 3% fish gelatine (Norland Products, Cranbury, NJ) in washing buffer (50 mM Tris-HCl, 150 mM NaCl, 0.2 % Tween 20). Blocking step and subsequent incubation with samples and standards followed by detection with antibodies were performed for 1h at 37 °C. Serial dilutions of ICS#2 (International Complement Standard #2, a pool of sera from healthy volunteers activated with aggregated IgG and zymosan (Bergseth et al., 2013) ) were used as a standard. Since ICS#2 is a source of every possible complement activation product, their content was defined as 1000 complement activation units (CAU) per one millilitre of ICS#2. Detection was performed by anti C4d #253 Ab (Quidel) diluted 1:1500 in PBS + 0.2% Tween 20 followed by goat anti-mouse, HRP-conjugated Ab (Dako, Glostrup, Denmark) diluted 1:1000. Assay was developed with OPD tablets (Dako) according to the manufacturer's instruction and absorbance at 490 nm was measured using Cary50 MPR microplate reader (Varian, Palo Alto, CA).
10
Enzyme-linked Immunosorbent Assay for Lipocalin-2
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Supernatants were collected and clarified by centrifugation. A total of 50 μl of each sample were applied to an ELISA 96-well-plate previously covered with a detection antimouse Lipocalin-2/NGAL monoclonal antibody (R&D) and blocked for 1 h. After sample incubation, biotinylated anti-mouse Lipocalin-2/NGAL detection antibody (R&D) was added. Afterwards, HRP-conjugated avidin (Invitrogen) was added for 1 h. Finally, colour reagent (OPD tablets, Dako, Glostrup, Denmark) was added and colour development was assessed by a microplate reader. Total protein amount in the sample was determined by the Lowry method for calculating the Lcn-2 amount per mg of total protein.
For cytokine levels of TNF-α and IL-10, supernatants were processed according to manufacturer's kit instructions (Endogen, Pierce Biotechnology, Barcelona, Spain).
Small interfering RNA (siRNA) transfections siRNA transfection was performed as described previously [9] . Briefly, siRNA oligonucleotides were delivered to primary macrophages using Lipofectamine 2000 according to manufacturer's recommended protocol (Invitrogen). We used three different siRNA clones to test the knockdown efficiency. For further experiments, we applied siRNA clone 3. Lcn-2 and megalin gene expression were measured by real-time qRT-PCR.
For cytokine levels of TNF-α and IL-10, supernatants were processed according to manufacturer's kit instructions (Endogen, Pierce Biotechnology, Barcelona, Spain).
Small interfering RNA (siRNA) transfections siRNA transfection was performed as described previously [9] . Briefly, siRNA oligonucleotides were delivered to primary macrophages using Lipofectamine 2000 according to manufacturer's recommended protocol (Invitrogen). We used three different siRNA clones to test the knockdown efficiency. For further experiments, we applied siRNA clone 3. Lcn-2 and megalin gene expression were measured by real-time qRT-PCR.
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