To quantify TCC deposition on cultured and stimulated human IVD cells, a cell-based ELISA was used [27 (link)]. 6000 cells/well were seeded on a 96-well plate and stimulated for 2 h as described previously. After stimulation, cells were fixed with 4% formalin for 15 min, and TCC deposition was detected as follows. Briefly, after a blocking step with 5% bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany) in DPBS at 37 °C for 1 h, cells were incubated for 2 h at 37 °C with an anti-C5b-9/TCC antibody (1:4000 dilution, Abcam, Cambridge, UK) and subsequently with an anti-rabbit IgG HRP conjugated antibody (1:10,000 dilution, Sigma-Aldrich, Taufkirchen, Germany) for 1 h at room temperature. Afterward, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Taufkirchen, Germany) was added for 20 min at room temperature. Finally, substrate conversion was stopped with stop solution (R&D Systems, Minneapolis, MN, USA), and absorbance was determined at 450 nm (microplate reader, infinite M200 pro, Tecan, Crailsheim, Germany).
Anti c5b 9 tcc antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-C5b-9/TCC antibody is a laboratory reagent that specifically binds to the C5b-9 complex, also known as the terminal complement complex (TCC). The C5b-9 complex is formed during the terminal phase of the complement system activation and is involved in cell lysis. This antibody can be used to detect and quantify the presence of the C5b-9 complex in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Stop solution, by R&D Systems (2 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Light microscopy, by Zeiss (1 mentions)
5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Anti rabbit igg peroxidase, by Merck Group (1 mentions)
2 protocols using anti c5b 9 tcc antibody
1
Quantifying Terminal Complement Complex Deposition
2
Quantifying Terminal Complement Complex (TCC) in Cells
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For quantification of TCC deposition in in vitro cell cultures, a cell-based ELISA was used [17] (link). Briefly, after a blocking step with 5% bovine serum albumin (Sigma-Aldrich) in DPBS at 37 °C for 1 h, cells were incubated for 2 h at 37 °C with anti-C5b-9/TCC antibody (1:4000 dilution, Abcam). Anti-rabbit IgG peroxidase (1:10,000 dilution, Sigma-Aldrich) was used as secondary antibody. After 1 h of incubation at room temperature with the secondary antibody, cells were incubated with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich) for 15 min at room temperature. Before the addition of stop solution (R&D Systems), 50 µL of the TMB solution was transferred to a new plate, to prevent cell lysis by the sulfuric acid. Absorbance values were measured at 450 nm. The cells were washed twice with PBS and imaged with light microscopy (Zeiss). DNA concentration was determined using the Quant-iT PicoGreen doublestandard DNA kit (Invitrogen), following the manufacturer's instructions.
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