For flow cytometry the following antibodies were used: PE-conjugated mouse anti- HLA-ABC (1:100, 555553, BD Biosciences), human anti- HLA-A2 antibody (3 µg mL-1, SN607D8), mouse anti-human CD55 (1:500, ab1422, Abcam), mouse anti-C3 (1:1000, RFK22, in-house generated), mouse anti-C5b-9 (1:100, AE11, Hycult Biotech), PE-conjugated mouse anti-human CD46 (1:100, 12-0469-42, Invitrogen), and APC-conjugated mouse anti-human CD59 (1:100, 17-0596-42, Invitrogen) were used as primary antibodies. Secondary antibodies used for detection were PE-conjugated goat anti-human IgG (1:1000, 109-116-098, Jackson) and PE-conjugated goat anti-mouse (1:100, R0480, DAKO). All antibody incubations were done for 30 min on ice with subsequent washing in PBS + 0,1% BSA. Flow cytometry was performed on a LSR-II (BD) and acquired results analyzed using FlowJo (BD).
R0480
Manufactured by Agilent Technologies
Sourced in United States
The R0480 is a UV-Vis spectrophotometer designed for laboratory use. It measures the absorbance or transmittance of light in the ultraviolet and visible light spectrum. The core function of the R0480 is to quantify the concentration of substances in a sample by analyzing the light absorption properties of the sample.
Automatically generated - may contain errors
5 protocols using r0480
1
Flow Cytometry for Cell Surface Markers
2
ET(B)R and IE Expression by Flow Cytometry
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Flow cytometry was performed as described previously [8 (link)]. Single-color staining was performed using primary mouse anti-ETBR (1:200; a gift from Dr. Tomoko Doi, Kyoto University) [25 (link)] or mouse anti-IE (1:300) (MAB810R; Millipore). For dual staining, cells were incubated with rabbit anti-ETBR (1:200) (AER-002; Alomone Labs) for 30 minutes at room temperature and then washed and fixed with Reagent A followed by permeabilization with Reagent B (Molecular Probes; Invitrogen, Life Technologies) along with primary mouse anti-IE according to the manufacturer's protocol. The positivity was revealed using secondary antibody swine anti-rabbit (1:100) (F0054; Dako) conjugated to fluorescein isothiocyanate isomer 1 or goat anti-mouse (1:100) (R0480; Dako) conjugated to R-phycoerythrin (RPE).
3
Properdin Binding and C3 Deposition
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properdin binding, using either purified or oligomeric forms of properdin (Quidel), was performed as described above. Cells were washed and incubated (30 min, 37˚C) with 10% NHS either diluted in RPMI, RPMI‐MgEGTA (RPMI‐1640 containing 10 mM EGTA and 5 mM MgCl2), or in RPMI‐EDTA (RPMI‐1640 containing 10 mM EDTA). Cells were washed and incubated (30 min, 4˚C) with mouse‐anti human C3 (1/600, RFK22, in‐house generated), followed by incubation with goat anti mouse–PE(F(ab’)2 goat anti‐mouse‐IgG‐RPE) (5 μg/mL, R0480, DAKO) diluted in RPMI (no phenol red, 30 min at 4˚C). C3 deposition was determined by flow cytometry (LSR‐II, BD).
4
Flow Cytometric Analysis of Complement Deposition
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Cells were blocked with pooled heat‐inactivated NHS (∆NHS, 10%) for 15 min at room temperature. Next, cells were washed and incubated with properdin (Quidel). Cells were washed and incubated with 10% NHS diluted in the described complement buffers (30 min, 37˚C). Cells were incubated with mouse monoclonal anti human C5b‐9 (1 μg/mL, AE11, Hycult Biotech; 30 min, 4˚C) followed by incubation with goat anti mouse–PE(F(ab’)2 goat anti‐mouse‐IgG‐RPE) (5 μg/mL, R0480, DAKO), diluted in RPMI (no phenol red, 30 min at 4˚C). Deposition was determined by flow cytometry.
5
Properdin Binding Assay on Cells
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Purified properdin, either from Quidel (A412) or CompTech (A139, Tyler, TX, USA) was aliquoted and kept frozen (−80˚C). Recombinant produced properdin was stored at 4˚C. A total of 10 μg/mL properdin (Quidel; or otherwise indicated at the figure legends), was diluted in RPMI‐1640 medium (no phenol red), followed by incubation with the cells for 1 h at 4˚C. Cells were washed and incubated with mouse‐monoclonal anti‐human properdin (2 μg/mL, A233, Quidel) for 30 min at 4˚C, followed by incubation with goat anti‐mouse immunoglobulins/RPE, goat F(ab’)2, RPE (5 μg/mL, R0480, DAKO, Santa Clara, CA, USA) for 30 min at 4˚C. Binding of oligomeric properdin structures P2, P3, and P4 was examined following a similar protocol. Binding was assessed using flow cytometry (LSR‐II, BD).
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