properdin binding from NHS was examined by incubating necrotic Jurkat T cells with different percentages of NHS. Binding was examined as described above. In addition, properdin (10 μg/mL, Quidel) was incubated with various percentages of ∆NHS (twofold dilutions starting from 50%) for 30 min at 4˚C. Next, the mixture was added to necrotic Jurkat T cells and incubated for 1 h at 4˚C. Cells were washed and properdin binding was determined as described above. For C3 deposition, cells were washed with RPMI‐MgEGTA and incubated with 10% properdin‐depleted serum (A339, Comptech) for 30 min at 4˚C. Cells were washed and C3 deposition was determined by flow cytometry, as described above.
Properdin
Manufactured by Quidel
Sourced in Hungary
Properdin is a laboratory equipment product manufactured by Quidel. It is a protein that plays a role in the activation of the alternative complement pathway, which is part of the body's immune response. Properdin serves as a positive regulator of this pathway.
Automatically generated - may contain errors
5 protocols using properdin
1
Properdin Binding and C3 Deposition
2
Complement pathway protein detection
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Complement proteins FH (#341274), FB (#341262), C3b (#204860), C3 (#204885), factor I (FI) (#341280), C1q (#204876), goat anti-FB (#341272) and goat anti-FH (#341276) antiserum were obtained from Merck (Darmstadt, Germany; obtained via Merck Life Science Kft., Budapest, Hungary). FD (#A409), properdin (#A412), mouse anti-C5b-9 (#A239), anti-FB (Bb) (#A227), anti-FB (Ba) (#A225) and C3a EIA kit (#A032) were from Quidel (obtained via Biomedica, Budapest, Hungary). Purified human IgG (#I2511), human serum albumin (#A3782), human alpha-1 antitrypsin (#SRP6312), antibodies against C1q (#234390), human IgG (#A6029), IgM (#A0420), IgA (#A0295), IgG1 (clone HP-6001, #I9388), IgG2 (clone HP-6002, #I9513), IgG3 (clone HP-6050, #I7260), IgG4 (clone HP-6025, #I7385), IgGκ (clone KP-53, #K4377), IgGλ (clone HP-6054, #L6522), and avidin-HRP (#A7419) were purchased from Merck. Mouse IgG1 (clone MOPC-21, #BZ-400101) was obtained from BioLegend (San Diego, CA). MBL (#9086-MB) and biotin-conjugated goat anti-MBL (#BAF2307) were from R&D Systems (Minneapolis, MN), HRP-conjugated goat anti-mouse (#P0447), rabbit anti-goat (#P0449) and swine anti-rabbit (#P0217) antibodies were from DAKO (Hamburg, Germany). Goat anti-C3 F(ab’)2 (#55062) and HRP-conjugated goat anti-C3 F(ab’)2 (#55237) were from MP Biomedicals (Solon, OH).
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 and Salp20 Binding Interactions
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A total of 10 μg/mL properdin (Quidel) was preincubated with various concentrations of Salp20 (30 min, 4˚C). Next, cells were incubated with the mixture (1 h, 4˚C). properdin binding was analyzed by flow cytometry as described above. A FITC‐labeled form of Salp20 was used to investigate the direct interaction of Salp20 with the cell surface.
The effect of Salp20 on properdin binding and AP activation was examined. properdin and Salp20 (1000 nM) were preincubated and the mixture was added to necrotic Jurkat T cells. Cells were washed and incubated with 10% NHS diluted in RPMI‐MgEGTA (30 min, 37˚C). C3 deposition was determined as described above.
The effect of Salp20 on properdin binding and AP activation was examined. properdin and Salp20 (1000 nM) were preincubated and the mixture was added to necrotic Jurkat T cells. Cells were washed and incubated with 10% NHS diluted in RPMI‐MgEGTA (30 min, 37˚C). C3 deposition was determined as described above.
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