Cerebellum tissues were homogenized by sonication in 0.2 M perchloric acid (100 μM EDTA•2Na) and centrifuged at 20.000×g for 15 min. Supernatants were kept frozen until analysis. Tissue glutamate concentrations were measured by enzyme-linked immunoassay (ELISA). Ninety-six well plates were coated with cerebellum homogenates (10 μg/well) at 4°C overnight. After washing with PBS (phosphate buffered saline, 0.05% Tween 20), in each well, anti-glutamate antibody was added (rabbit, 1:1,000, ab37070, Abcam) and incubated for 2 hours at room temperature. Thereafter, the samples were treated with horseradish peroxidase-conjugated anti-rabbit antibody (1:1,000, Sigma-Aldrich) for 2 hours at room temperature and with substrate solution (R&D systems, Minneapolis, MN, USA) for 20 min. The optical density was measured at 450 nm with a micro-plate reader (SpectraMAX M5, molecular device, Sunnyvale, CA, USA) after stopping peroxidase response with a stop solution (R&D systems).
Stop solution
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Stop solution is a laboratory reagent used to halt or terminate chemical reactions. It is a common component in various laboratory assays and protocols.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Streptavidin hrp, by R&D Systems (2 mentions)
Anti c5b 9 tcc antibody, by Abcam (2 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Merck Group (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Anti gal 3 antibody, by BioLegend (1 mentions)
Streptavidin horseradish peroxidase conjugate, by R&D Systems (1 mentions)
Biotinylated vegf a165, by R&D Systems (1 mentions)
Mab1937, by Merck Group (1 mentions)
Hrp goat antimouse, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Human vegf elisa kit, by R&D Systems (1 mentions)
Biotinylated horse antirabbit secondary antibody, by Vector Laboratories (1 mentions)
Hrp substrate, by R&D Systems (1 mentions)
Anti fn antibody, by Merck Group (1 mentions)
Synergy 2 microplate reader, by Agilent Technologies (1 mentions)
Startingblock t20 blocking buffer, by Thermo Fisher Scientific (1 mentions)
Dnase, by Merck Group (1 mentions)
Protein free blocking buffer, by Thermo Fisher Scientific (1 mentions)
96 well microplate, by Thermo Fisher Scientific (1 mentions)
21 protocols using stop solution
1
Cerebellum Glutamate Quantification by ELISA
2
Binding Affinity of Galectin-3 to Bacterial LPS
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Experiments was performed as described previously, with slight modifications36 (link). The 96-well ELISA plates (Costar High binding 3590, Fisher Scientific, Japan) were coated with Aggregatibacter actinomycetemcomitans-LPS (A.a.-LPS) kindly provided by Dr. Tatsuji Nishihara of Kyushu Dental College, Japan), P.g.-LPS, Escherichia coli-LPS (E. coli-LPS; Wako Pure Chemical Industries, Ltd., Ohsaka, Japan) (50 μg/ml each), or 3% bovine serum albumin (BSA; as a control) and incubated overnight at 4 °C. Plates were washed twice with ELISA buffer (Tris-buffered saline with 0.05% Tween 20, 1 mM CaCl2, and 0.1% BSA, pH 7.4), and 3% Gelatin was used for blocking at 37 °C for 1 h. rhGal-3 was added to each well at different final concentrations (0, 0.05, 0.15, 0.6, or 2 μg/ml) and incubated overnight at 4 °C. An anti-Gal-3 antibody (BioLegend, Inc.) was also added to each well, and the plates were incubated overnight at 4 °C. For detection, a horseradish peroxidase-conjugated anti-rat IgG (GE Healthcare Life Sciences) was added, and tetramethyl benzidine substrate (colour reagent A & B, 1: 1, R&D Systems) was used for visualization. After 20 min, stop solution (R&D Systems) was added. O.D.s were measured at 450 nm.
3
NRP-1 Binding Assay with VEGF-A
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The surface of microplates (Corning Inc., Corning, NY, USA) was coated overnight at RT with NRP-1 at a concentration of 2 µg/mL (R&D Systems, Inc., Minneapolis, MN, USA) in PBS. A solution of PBS containing 0.5% bovine serum albumin (BSA; blocking buffer) was then used to block the plates, to prevent nonspecific interactions, during 1 h at 37°C. In all, 5 ng/mL of biotinylated VEGF-A165 (R&D Systems, Inc.) in blocking buffer containing 2 µg/mL of heparin was used to assess the binding of compounds to NRP-1. Biotinylated VEGF-A165 was added to the coated wells in competition with the different compounds or excess of non-labeled VEGF-A165, as a positive control. The plates were washed after a 2 h incubation at RT, and the amount of bound biotinylated VEGF-A165 was stained with streptavidin horseradish peroxidase conjugate (R&D Systems, Inc.). The reaction was stopped after 20 min at RT by the addition of Stop Solution (R&D Systems, Inc.). Optical densities were measured at 450 nm. Results were expressed as relative absorbance to wells containing only blocking buffer. Three wells per condition were used.
4
Fibronectin Adsorption and Antibody Detection
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Enzyme-linked immunosorbent assay (ELISA) was performed on PEA and PMA coated with different concentrations of FN using primary monoclonal antibodies HFN7.1 (DSHB, Iowa City, IA) and mAb1937 (Millipore) combined with HRP-Goat Anti-Mouse (Invitrogen, Carlsbad, CA) secondary antibody, which is coupled with a peroxidase for absorbance reading. FN was adsorbed at 20 and 1 μg/mL as well as a control without protein on PEA and PMA for 1 h. Samples were washed with PBS, transferred to new 24-well plates, and then blocked with PBS/BSA 1% w/v for 30 min at room temperature. Subsequently, samples were incubated with the primary antibodies for 1 h at room temperature and washed with PBS/Tween-20 0.5% v/v. The secondary antibody (HRP-Goat Anti-Mouse) was then added on all samples for 1 h at room temperature in the dark and washed with PBS/Tween-20 0.5% v/v. The substrate (color reagents A and B from R&D Systems, Minneapolis, MN) was then added for 20 min at room temperature, in the dark, stopped with a stop solution (R&D Systems), and transferred to a 96-well plate and absorbance was read at 450 and 550 nm.
5
VEGF Secretion Quantification by ELISA
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The above collected supernatants were used for VEGF secreted protein detection. The secreted levels of VEGF were quantified using corresponding Human VEGF ELISA kits (DY293B; R&D Systems, Inc.). Briefly, supernatants were centrifuged at 3,500 x g for 5 min, at 4°C to remove any impurity. Microplate strips were removed from the plate frame. Assay Diluent RD1W (50 μl) (R&D Systems, Inc.) was added to each well. Then, 200 μl of VEGF standards, controls, or samples were added to each well. Wells were covered with a plate sealer and incubated at room temperature for 2 h. After 3 washes, 200 μl VEGF conjugate was added and incubated at room temperature for 20 min in the dark followed by adding 50 μl of Stop Solution (R&D Systems, Inc.) to each well. Light absorption values were read within 30 min using a microplate reader (Molecular Devices, San Jose, USA) (450 nm).
6
Quantifying Integrin-Binding Protein Availability
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The availability of
FN adsorbed on polymer surfaces as well as that of the RGD and synergy
domains was determined by ELISA. Samples were blocked for 30 min with
1% w/v BSA (Sigma-Aldrich). To determine the availability of FN, we
incubated rabbit polyclonal anti-FN antibody (Sigma-Aldrich, 1:10 000)
for 1 h, followed by a 1 h incubation with biotinylated horse antirabbit
secondary antibody (Vectorlabs, 1:10 000), both at room temperature
(RT). Samples were then incubated with HRP-streptavidin (R&D Systems)
for 20 min, washed, and incubated with HRP substrate (R&D Systems)
for 20 min. After stopping the reaction, using the stop solution,
absorbance was measured at 450 (maximal absorbance of the tag) and
540 nm (blank control, to determine background absorbance). To assess
the availability of cell-binding domains, we incubated samples at
RT with monoclonal mouse primary antibody (mAb1937, 1:20,000 in 1%
BSA or HFN 7.1 antibody for the synergy or RGD domain, respectively).
The samples were then washed with 0.5% Tween 20 and incubated with
goat antimouse HRP-tagged secondary antibody (1:10 000 in 1%
BSA solution) for 1 h (RT). After washing with 0.5% Tween 20, samples
were incubated with HRP substrate solution in the absence of light
for 20 min. The reaction was terminated with stop solution (R&D
Systems) and absorbance was measured.
FN adsorbed on polymer surfaces as well as that of the RGD and synergy
domains was determined by ELISA. Samples were blocked for 30 min with
1% w/v BSA (Sigma-Aldrich). To determine the availability of FN, we
incubated rabbit polyclonal anti-FN antibody (Sigma-Aldrich, 1:10 000)
for 1 h, followed by a 1 h incubation with biotinylated horse antirabbit
secondary antibody (Vectorlabs, 1:10 000), both at room temperature
(RT). Samples were then incubated with HRP-streptavidin (R&D Systems)
for 20 min, washed, and incubated with HRP substrate (R&D Systems)
for 20 min. After stopping the reaction, using the stop solution,
absorbance was measured at 450 (maximal absorbance of the tag) and
540 nm (blank control, to determine background absorbance). To assess
the availability of cell-binding domains, we incubated samples at
RT with monoclonal mouse primary antibody (mAb1937, 1:20,000 in 1%
BSA or HFN 7.1 antibody for the synergy or RGD domain, respectively).
The samples were then washed with 0.5% Tween 20 and incubated with
goat antimouse HRP-tagged secondary antibody (1:10 000 in 1%
BSA solution) for 1 h (RT). After washing with 0.5% Tween 20, samples
were incubated with HRP substrate solution in the absence of light
for 20 min. The reaction was terminated with stop solution (R&D
Systems) and absorbance was measured.
7
SARS-CoV-2 Antibody Detection by ELISA
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For the indirect ELISA, a 96-well plate was coated with 2 μg/ml of the S1, RBD, and N proteins (Sino Biological, China) diluted in phosphate-buffered saline (PBS) overnight at 25°C. Each well was blocked with 300 μL of StartingBlock T20 blocking buffer (Thermo Fischer Scientific, Waltham, MA, USA) for 1 h at 25°C. Heat-inactivated convalescent-phase serum samples from COVID-19 patients and healthy donors were diluted in blocking buffer at a ratio of 1:200, and then 100 μL of the samples was added to the 96-well plate in duplicate. After the plates were washed, horseradish peroxidase (HRP)-tagged anti-human (IgG, IgM, and IgA) antibodies (Abcam, Cambridge, UK) were diluted 1:10,000 with blocking buffer and added to the wells (100 μL/well), and the plate was then incubated for 1 h at 25°C. Samples with N antibodies were incubated only for 30 min at 25°C because of the higher signal. The chromogenic reagent 3,3′,5,5′-tetramethylbenzidine (TMB) was mixed with an equal volume of color A and color B (R&D Systems, Minneapolis, MN, USA). The TMB reaction time for the S1 and RBD ELISA was 5 min, whereas that for the N protein ELISA was 10 min. After the reaction, stop solution (R&D Systems) was added to the wells and the OD was measured immediately at 450 nm using a Synergy 2 microplate reader (Bio-Tek, Winooski, VT, USA).
8
Quantification of Citrullinated Histone H3 in Serum
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To quantify CitH3 in the blood, we developed a “sandwich” indirect ELISA. In brief, 0.2ug/well anti-CitH3 monoclonal antibody was coated onto 96-well plate as capture antibody overnight at 4 °C, then plates were blocked by protein-free blocking buffer (Thermo Scientific, Rockford, IL, USA) for 2 h at room temperature (RT). Serum was treated with DNase (150 unit/ml, Sigma Aldrich, St. Louis, MO, USA) for 1 hour at 37 °C with supplement of 1 mM calcium chloride. DNase treated serum (20 µl) was added to the wells with 80 µl blocking buffer and incubated at RT for 2 hours. After four washings, anti-CitH3 rabbit polyclonal antibody (1:3000 diluted, Abcam, Cambridge, MA, USA) was added as detecting antibody for 2 hours at RT. Following 4 times of washing, anti-rabbit peroxidase-labeled secondary antibody (1:50000 diluted, Jackson ImmunoResearch, West Grouve, PA, USA) was incubated in wells for 1 hour at RT. After 4 thorough washes to remove extra secondary antibodies, plate was developed with 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) for 20 minutes in dark followed by stop solution (R&D Systems Inc., Minneapolis, MN, USA). Absorbance at 450 nm wavelength was determined. Synthesized CitH3 peptide (New England peptide, Gardner, MA, USA) was utilized to generate the standard curve.
9
ELISA Assay for LDHA Binding
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ELISA was performed using 96-well microplates (Thermo Fisher Scientific) that were coated with 2 μg/ml of BSA or vimentin. The wells were incubated with 5% (w/v) BSA-PBS 1 h at room temperature. The wells then incubated with recombinant mouse LDHA (160–0 ng/ml) or BSA (160–0 ng/ml) diluted in PBST. Two hours after incubation, the wells were washed and anti-LDHA antibody was added. One hour after incubation, the wells were incubated with HRP-conjugated secondary antibodies. Protein bindings were detected by using substrate reagent, and stop solution (R & D Systems). Absorbance was measured at 450 nm.
10
Quantifying PD-1/PD-L1 Binding Interactions
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Streptavidin-HRP (cat# DY998), substrate reagent pack (cat# DY999), and stop solution (cat# DY994) were purchased from R&D Systems (Minneapolis, MN). Ninety-six-well plates were coated with 100 ng of human PD-L1 ECD (cat# FCL0784, G&P Biosciences) or mouse PD-L1 ECD (cat# FCL3502, G&P Biosciences) overnight at 4°C and blocked with 2% BSA for 2 h at room temperature. dAbs were loaded in each well and incubated for 1 h at room temperature. Next, unbound dAbs were removed by washing with PBS. Five hundred nanograms of biotinylated human PD-1 ECD (cat# FCL0761B, G&P Biosciences) or mouse PD-1 (cat# FCL1846B, G&P Biosciences) was loaded in each well and incubated for 1 h. After washing with PBS, 100 µl of Streptavidin-HRP (diluted 1:200 in 1% BSA) was added and incubated for 20 min, followed by the addition of substrate reagents and stop solution. The absorbance at 450 nm was recorded with the reference wavelength at 540 nm.
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