3 3 5 5 tetramethylbenzidine substrate

HUVECs were seeded in 96-well plates at a cell density of 5 × 10⁴/well and grown to confluence for 72 h with daily medium change. HUVECs were serum starved for 1 h before secretagogue treatment in serum-free M199 plus 10 mM Hepes. All reagents were brought to room temperature before the experiment. After respective treatments were terminated, cells were washed with 1× PBS and incubated with 4% PFA for 5 min, followed by a blocking step with BSA. Incubation with P-selectin primary antibody (Santa Cruz; sc-19672) and HRP-conjugated secondary antibody (Jackson ImmunoResearch Labs; 115-035-003) solution was performed at room temperature. 3,3′,5,5′-Tetramethylbenzidine substrate (Sigma-Aldrich; T0440) was added for 5 min with gentle agitation until the characteristic color change was obtained. The reaction was terminated with an equal amount of 0.5 M H₂SO₄ and read at 450 nm on the Flexstation II³⁸⁴.

Remaining blood serum samples from 41 dogs (18 controls, 13 lymphomas and 10 during CHOP therapy), referred to the “NeoVet” veterinary clinic for periodic blood checks, and blood sera of NOD-SCID mice bearing CLBL1-Luc tumors were used for the detection of soluble DLA-DR levels. In accordance with the provisions of the Act of January 15, 2015, item 266 on the protection of animals used for scientific and educational purposes, the use of blood samples of dogs for clinical veterinary research does not require the consent of local ethics committees.
96 well plates (Nunc) were coated with B5 mAb in PBS (1 µg/mL) overnight at 4 °C. On the next day, the plates were blocked with 5% non-fat milk for 1 h at room temperature (RT), then 20 times diluted blood sera of canine tumor-bearing mice or canine patients were incubated in a 0.5% milk solution at RT for 1 h. Next, biotinylated mAb E11 (1 µg/mL) was added to the solution and incubated for 1 h at RT, followed by the incubation with the Streptavidin-HRP conjugate (1:20,000); after final washes, the 3.3′5.5′-tetramethylbenzidine substrate (Sigma, St. Louis, MO, USA) was added for a 15 to 20-min incubation. The reaction was stopped with 1 N H₂SO₄. The absorbance was measured at 450 nm on a Wallac Victor plate reader (Perkin Elmer, Waltham, MA, USA). Each sample was prepared in triplicate.

The wells of a 96-well microtiter plate (Immulon 2, Dynatech Laboratories, Inc.) were coated overnight at 4 °C with 0.1 ml of 1 mg/ml rat tail collagen I (Corning) or mouse collagen IV (Corning) in 0.09% acetic acid. The wells were washed twice with 0.15 ml TBS and then blocked for 1 h at room temperature with 0.15 ml of 100 mg/ml bovine serum albumin (Sigma) in TBS. Recombinant GST-tagged I domains were serial diluted from 1 mM in various wash buffers (TBS containing 0.05% Tween-20, 10 mg/ml BSA, and either 5 mM EDTA, 1 mM CaCl₂, or 1 mM MgCl₂). The wells were washed once with 0.15 ml of the appropriate wash buffer, and then 0.1 ml of each I domain dilution was added and allowed to interact for 1.5 h at RT. Wells were washed three times with 0.15 ml of the appropriate wash buffer. Then 0.1 ml of a 1:1000 dilution of anti-GST HRP conjugate (GE Healthcare) in the appropriate wash buffer was added for 1 h at RT. Following incubation, the wells were washed three times, and then 0.06 ml of 3, 3′,5,5′-tetramethylbenzidine substrate (Sigma) was added for 1 h at RT. Reactions were stopped with 0.015 ml of 4 N H₂SO₄, and the plates were read at 450 nm. Representative binding plots are included in supplemental information (Fig. S8).

Receptor affinity was determined in a solid-phase direct virus-binding assay as previously described. In brief, influenza viruses were bound to fetuin-coated plates at 4 ˚C overnight. Biotinylated glycans (a2,3’SL; a2,6’SL; or a2,6’SLN; Glycotech Corporation, Gaithersburg, MD, USA) were added to influenza-coated plates at varying dilutions and incubated for 4 h. Glycan binding was detected by adding horseradish peroxidase-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA) followed by 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich, St Louis, MO, USA); the resulting absorbance at 450 nm was measured in a VICTOR3 1420 multilabel-counter plate reader (Perkin-Elmer, Waltham, MA, USA).

p24 ELISA was performed as described previously^{79 (link)}. Briefly, virus-pulsed cells and supernatant containing viral particles were lysed in ELISA lysis buffer (0.05% Tween 20, 0.5% Triton X-100, 0.5% casein in PBS). Anti-HIV-1CAp24 antibody (1:1000 or 1:2000; clone 183-H12-5C; NIH AIDS Research and Reference Reagent Program, Cat# 3537) was bound to Nunc MaxiSorp plates overnight. Lysed samples were captured for 2 h and incubated with biotinylated antibody to HIV-1CAp24 (1:2000 or 1:4000; clone 31-90-25; ATCC, Cat# HB-9725). 31-90-25 was biotinylated with the EZ-Link Micro Sulfo-NHS-Biotinylation Kit (Pierce). Samples were detected using streptavidin-HRP (Fitzgerald) and 3,3^′,5,5^′-tetramethylbenzidine substrate (Sigma-Aldrich). CAp24 concentrations were determined using recombinant HIV-1 IIIB p24 recombinant protein for standards (NIH AIDS Research and Reference Reagent Program).