The anti‐BSA ELISA was performed as previously described 1 . Briefly, 96‐well ELISA plates (Thermo Fisher Scientific) were coated with 1 µg BSA (Fraction V, Thermo Fisher Scientific) overnight at 4°C. Feline serum samples were diluted 1:5,000 along with negative and positive assay controls. Following sample incubation, diluted rabbit anti‐feline IgG H&L‐HRP (Southern Biotech, Birmingham, AL) was added to each well. TMB Peroxidase Substrate (KPL, Gaithersburg, MD) was added and samples were read on a microplate reader with Gen5 software (Biotek). Data is presented as a fold increase of color relative to the negative control for each patient.
Fraction 5
Manufactured by Thermo Fisher Scientific
Fraction V is a laboratory product manufactured by Thermo Fisher Scientific. It is a high-purity bovine serum albumin (BSA) used as a protein stabilizer and blocking agent in various biological and biochemical applications.
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Lab products found in correlation
D glucose, by Merck Group (4 mentions)
Fluospheres microspheres, by Thermo Fisher Scientific (3 mentions)
Crimson red fluorescent 625 645, by Thermo Fisher Scientific (2 mentions)
24 well plate, by Thermo Fisher Scientific (2 mentions)
96 well elisa plate, by Thermo Fisher Scientific (1 mentions)
Ultraview vox imaging system, by Yokogawa (1 mentions)
Csu 1 spinning disk, by Yokogawa (1 mentions)
Delta vision 3d, by Cytiva (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Standard cell scraper, by Corning (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Toluidine blue o, by Merck Group (1 mentions)
Trans blot system, by Bio-Rad (1 mentions)
Immobilon fl membrane, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Goat anti human ig hrp, by Southern Biotech (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Glutaraldehyde solution, by Merck Group (1 mentions)
Syringe pump, by KD Scientific (1 mentions)
10 protocols using fraction 5
1
Anti-BSA ELISA for Feline Serum
2
Immunofluorescence Staining of HUVECs
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Confluent HUVECs or iHUVECs were fixed in 2% paraformaldehyde for 20 min, washed three times with PBS, and blocked with blocking buffer (PBS with 5% BSA [Fraction V; Thermo Fisher Scientific] and 1% goat serum) was added for 30 min at RT. Cells were then incubated with primary antibody at 10 µg/ml in blocking buffer for 45 min at RT, washed extensively with PBS, and then incubated with secondary antibody at 4 µg/ml in blocking buffer for 45 min at RT protected from light. For the visualization of IQGAP1, cells were permeabilized before blocking for 4 min with 0.1% Triton X-100 (Sigma-Aldrich). Confocal images (all figures unless otherwise noted) were collected using an Ultraview VoX imaging system equipped with a Yokogawa CSU-1 spinning disk. Images were acquired with a 40× oil objective using Volocity software (numerical aperture [NA] 1.0). Widefield images (specifically Figs. 4 A and 5 C ) were collected using a restoration workstation (DeltaVision 3D; Applied Precision) equipped with an inverted microscope (model IX70; Olympus) using a 60× oil objective (NA 1.42). Some widefield images (specifically Fig. 4 A ) were processed using DeltaVision software and its associated constrained iterative deconvolution algorithm using a point spread function collected contemporaneously for each channel using standard fluorescence beads.
3
Phagocytosis Assay with Fluorescent Beads
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For the analysis of phagocytosis, leukocytes or RTS11 cells were seeded in 96-well plates (Nunc) at a cell density of 2×105 cells per well and incubated for 3 h at 20°C with fluorescent beads (FluoSpheres® Microspheres, 1.0 μm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 in the presence or absence of 100 ng/ml CK9. Cells were harvested using a standard cell scraper (Corning). Non-ingested beads were removed by centrifugation (100 x g for 10 min at 4°C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) D-glucose (Sigma). Cells were resuspended in staining buffer (PBS containing 1% FCS and 0.5% sodium azide), labelled with the flow cytometry antibodies when needed, and analyzed on a FACSCalibur flow cytometer.
4
Viral Infection and Quantification Assays
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Cells were infected by the addition of virus in a minimal volume at the appropriate dilution to give the indicated MOI for 1 hr at 37°C. After 1 hr cells were washed with PBS and replenished with fresh media without virus. Cells used to study IAV were cultured post-infection in DMEM, and cells used to study RSV were cultured post-infection in DMEM supplemented with 5% FBS. Supernatants were harvested at the indicated time post-infection and the viral titers were determined by plaque assay.
IAV plaque assays were performed with 10-fold serial dilutions of the virus samples on a confluent monolayer of MDCK cells,64 overlaid with MEM agarose overlay media containing 0.5% BSA (Fraction V, Fisher Scientific) and 1 μg/ml N-acetyl trypsin. Cells were incubated for 72 hr, and then plaques were fixed and visualized by staining with 0.1% toluidine blue O (Sigma). RSV immuno-plaque assays were performed with 2-fold serial dilutions of the virus samples on a confluent monolayer of HEp-2 cells. Cells were incubated for 24 hr in DMEM, and then they were probed with 1:200 biotinylated RSV antibody and stained with 1:500 ExtrAvidin Peroxidase, with visualization of plaques by the addition of 3-Amino-9-EthylCarbazole (AEC) substrate.
IAV plaque assays were performed with 10-fold serial dilutions of the virus samples on a confluent monolayer of MDCK cells,64 overlaid with MEM agarose overlay media containing 0.5% BSA (Fraction V, Fisher Scientific) and 1 μg/ml N-acetyl trypsin. Cells were incubated for 72 hr, and then plaques were fixed and visualized by staining with 0.1% toluidine blue O (Sigma). RSV immuno-plaque assays were performed with 2-fold serial dilutions of the virus samples on a confluent monolayer of HEp-2 cells. Cells were incubated for 24 hr in DMEM, and then they were probed with 1:200 biotinylated RSV antibody and stained with 1:500 ExtrAvidin Peroxidase, with visualization of plaques by the addition of 3-Amino-9-EthylCarbazole (AEC) substrate.
5
Quantifying Intestinal CD8+ DC Phagocytosis
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To analyze the phagocytic capacity of intestinal CD8+ DCs, leukocytes from the intestine were seeded in 24-well plates (Nunc) at a cell density of 1 × 106 cells per well and incubated for 16 h at 20 °C with fluorescent beads (FluoSpheres® Microspheres, 1.0 μm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 or without beads in the case of negative controls. After the incubation period, cells were harvested by gently pipetting and non-ingested beads were removed by centrifugation (100×g for 10 min at 4 °C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) d -glucose (Sigma). Cells were resuspended in L-15 with 5% FCS, labelled with the flow cytometry antibodies as described above and analyzed on a FACSCalibur flow cytometer. Flow cytometry analysis was performed using Flow Jo 10 software package.
6
Phagocytic Capacity of IgM+ B Cells
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To analyze the effect of CpGs on the phagocytic capacity of spleen and blood IgM+ B cells, spleen and blood leukocytes were seeded in 24-well plates at a cell density of 2 × 106 cells per well and incubated for 48 h at 20°C with the appropriate stimuli (5 μM CpG, 5 μM non-CpG, or media alone). After 48 h, the cells were collected and resuspended in L-15 medium without serum. The cells were then incubated for 3 h at 20°C with fluorescent beads (FluoSpheres® Microspheres, 1.0 μm, Crimson Red Fluorescent 625/645, 2% solids; Thermo Fisher Scientific) at a cell:bead ratio of 1:10 as described before (24 (link)). After the incubation period, cells were harvested by gently pipetting, and non-ingested beads were removed by centrifugation (100 × g for 10 min at 4°C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) D-glucose (Sigma). Cells were then resuspended in staining buffer, labeled with anti-IgM-FITC (1.14) (1 μg/ml) and analyzed on a FACS Calibur flow cytometer. In some experiments, cytochalasin B (0.05 μg/ml) was added to the cells immediately before the addition of the beads to verify active phagocytosis. Flow cytometry analysis was performed with FlowJo V10 software.
7
Immunoblotting for Protein Detection
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Cells were lysed with cell lysis buffer (300 mM NaCl, 50 mM Tris-HCl [pH 7.4], and 0.5% Triton X-100) containing protease (cOmplete, Roche) and phosphatase inhibitors (PhosStop, Roche), spun, and supernatant was collected. Total cell lysates were separated by SDS-PAGE and transferred to Immobilon-FL membranes (Millipore) using a Trans-Blot System (Bio-Rad). Membranes were blocked in Tris-buffered saline containing 0.5% Tween 20 and 5% BSA (Fraction V, Fisher Scientific) for 2 hr at room temperature. Primary antibody binding was achieved overnight at 4°C, whereas far-red fluorescent secondary antibody binding was achieved in 1 hr at room temperature. Odyssey (LI-COR Biosciences) was used for visualization.
8
Assessing Gill CD8+ DC Phagocytosis
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To analyze the phagocytic capacity of gill CD8+ DCs, gill leukocytes were seeded in 24-well plates (Nunc) at a cell density of 1 × 106 cells/well and incubated for 16 h at 20°C with fluorescent beads (FluoSpheres® Microspheres, 1.0 µm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 or without beads in the case of negative controls. After the incubation period, cells were harvested by gently pipetting, and non-ingested beads were removed by centrifugation (100 × g for 10 min at 4°C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) d -glucose (Sigma). Cells were resuspended in L-15 with 5% FCS, labeled with the flow cytometry antibodies and analyzed on a FACSCalibur flow cytometer or under the confocal microscope. In some experiments, cytochalasin B (0.05 µg/ml) was added to the cells immediately before the addition of the beads to verify active phagocytosis.
9
Measuring Anti-dsDNA and Anti-Sm/RNP Autoantibodies in SLE
10
Modified Desolvation Synthesis of Fluorescent BSA NPs
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NPs were synthesized by modified desolvation methods.24 (link) Briefly, bovine serum albumin (BSA, Fraction V, Fisher Scientific) was dissolved in 10 mM sodium chloride solution to make a 1.5% (w/v) BSA solution. The pH of the solution was adjusted to 9.0 with sodium hydroxide. The desolvation agent was a mixture of methanol and ethanol at the ratio of 7:3 (v/v). Then, 4 mL of the desolvation agent was added into 1 mL BSA solution using a syringe pump (KD Scientific) at 1 mL/min under constant stirring. Subsequently, 8% glutaraldehyde solution (Sigma-Aldrich) was added to the system to induce particle cross-linking. Cross-linking process was allowed under stirring at room temperature for 12 hrs. The synthesized NPs were washed with water for three times, using centrifugal filter membrane units (molecular weight cutoff 100 kDa, Amicon). Fluorescent fluorescein isothiocyanate (FITC, ThermoFisher Scientific) and Cyanine 7 (Cy7, Lumiprobe) was conjugated to monomeric BSA according to the manufacturer’s protocol, respectively. Fluorescently labeled nanoparticles (FITC-NP and Cy7-NP) were synthesized by the same procedures as described above using FITC-BSA or Cy7-BSA instead of BSA.
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