CXCL12 and MIF secretion in CM was measured using the Human CXCL12/SDF-1 DuoSet ELISA and MIF Quantikine ELISA kits (R&D Systems) following the manufacturer’s protocols. The absorbance was measured at 450 nm and corrected at 540 nm on a microplate reader Infinite® 200 PRO (Tecan, Switzerland).
Human cxcl12 sdf 1 duoset elisa
Manufactured by R&D Systems
The Human CXCL12/SDF-1 DuoSet ELISA is a collection of components used for the quantitative determination of human CXCL12/SDF-1 concentrations in cell culture supernates, cell lysates, serum, and plasma samples. It includes a capture antibody, a detection antibody, and a standard.
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3 protocols using human cxcl12 sdf 1 duoset elisa
1
Quantification of CXCL12 and MIF Secretion
2
Radiation-Induced SDF-1 Release Assay
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T98G cells (250.000) were seeded in 75 cm2 cell culture flasks in RPMI-1640 medium containing 10% FCS and grown over night. Cells were washed with PBS and serum depleted for 24 h. Thereafter, cells were washed with PBS and overlaid with NaCl solution (see patch-clamp section) containing protease inhibitors (Roche, cOmplete Mini, EDTA-free, #04693159001). After 30 min incubation, cells were irradiated with 0 or 2 Gy. After further 2 h the medium was harvested and the SDF-1 concentration determined using an ELISA assay kit (R&D Systems, Human CXCL12/SDF-1 DuoSet ELISA, #DY350).
3
Sustained Release of CXCL12 from Peptide-Coated Scaffolds
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Coating with peptide, heparin and CXCL12 was performed as described above. Scaffolds were placed in reaction tubes filled with 300 µl PBS and incubated at 37 °C and shaking for the respective time points. After 3, 6, 9, 24, 48 and 72 h, the supernatant was collected and replaced with fresh PBS. The amount of CXCL12 present in each fraction was determined using the quantification kit "Human CXCL12/SDF-1 DuoSet ELISA" in combination with the "DuoSet Ancillary Reagent Kit" (both R&D Systems) according to the manufacturer's protocol. The data is represented as cumulative amounts released over time (mean ± sem) from three independent experiments performed in triplicates.
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