For binding assay, breast cancer cells were untreated (-) or treated with BRL 10 μM in phenol red-free media containing 5% CT-FBS for 24 h. Then, cells were harvested with versene reagent, washed twice in PBS and 103 cells/well were incubated with CAF-CM in a final volume of 100 μl binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 150 mM NaCl, 5 mM MgCl2, 5% bovine serum albumin). Samples were incubated for 60 min at 4°C with rotation. After incubation, cells were centrifuged and washed twice with 300 μl wash buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 500 mM NaCl, 5 mM MgCl2) and freezed to −20°C and thawed to room temperature 3 times and then centrifuged at 1500×g for 10 minutes at 2 − 8°C to remove cellular debris. The supernatants were collected for assaying human SDF-1α levels (R&D Systems). The optical density of each well was determined using a microplate reader at 450 nm (Bio-Rad Model 3550 microplate reader, Richmond, CA) and normalized for cell number. At least three independent experiments were performed.
Human cxcl12 sdf 1 alpha quantikine elisa kit
The Human CXCL12/SDF-1 alpha Quantikine ELISA Kit is a quantitative sandwich enzyme immunoassay designed to measure human CXCL12/SDF-1 alpha levels in cell culture supernates, serum, and plasma.
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8 protocols using human cxcl12 sdf 1 alpha quantikine elisa kit
SDF-1α Quantification and Binding Assay
For binding assay, breast cancer cells were untreated (-) or treated with BRL 10 μM in phenol red-free media containing 5% CT-FBS for 24 h. Then, cells were harvested with versene reagent, washed twice in PBS and 103 cells/well were incubated with CAF-CM in a final volume of 100 μl binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 150 mM NaCl, 5 mM MgCl2, 5% bovine serum albumin). Samples were incubated for 60 min at 4°C with rotation. After incubation, cells were centrifuged and washed twice with 300 μl wash buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 500 mM NaCl, 5 mM MgCl2) and freezed to −20°C and thawed to room temperature 3 times and then centrifuged at 1500×g for 10 minutes at 2 − 8°C to remove cellular debris. The supernatants were collected for assaying human SDF-1α levels (R&D Systems). The optical density of each well was determined using a microplate reader at 450 nm (Bio-Rad Model 3550 microplate reader, Richmond, CA) and normalized for cell number. At least three independent experiments were performed.
Cytokine and Chemokine Profiling of MSPCs and JMML Cells
Quantification of Plasma Biomarkers
Serum levels of extended range C-reactive protein were determined using the particle-enhanced turbidimetric immunoassay (PETIA technique; Siemens Dimension, USA). The reference range in this method is 0.0–5.0 mg/l. BNP levels were assessed using an Alere Triage reagent pack (Alere Inc., Ottawa, ON, CAN) and analysed in an automated D × I 800 immunoanalyzer (Beckman-Coulter, Fullerton, CA, USA).
Total GLP-1 was measured by a magnetic bead kit (Merck-Millipore, Vimodrone, Italy) according to kit instructions and analyzed on a MagPix (Luminex Corporation). The assay is based on the attachment of GLP-1 to magnetic beads and processing using LED excitation.
CXCL12 Secretion Quantification in Cell Lines
Biomarker Measurement Protocol for Plasma
As Level 1 outcomes, we obtained peripheral BDNF levels using an ELISA kit (Human Free BDNF Quantikine ELISA Kit; R&D Systems, Minnesota, USA). The rest of the molecular markers were selected according to the Projecte Moviment trial (Castells-Sánchez et al., 2019 (link)). TNF-α, ICAM-1, HGF, and SDF1-α levels were analyzed quantitatively using the corresponding ELISA immunoassay method (Human TNF-α Quantikine HS ELISA, Human ICAM-1/CD54 Allele-specific Quantikine ELISA Kit, Human HGF Quantikine ELISA Kit, Human CXCL12/SDF-1 alpha Quantikine ELISA Kit; R&D Systems, Minnesota, USA).
Quantifying Plasma Biomarkers in Healthy Adults
We obtained peripheral BDNF levels using an ELISA kit (Human Free BDNF Quantikine ELISA Kit; R&D Systems, Minnesota, United States). The Proteome Profiler Human™ XL Cytokine Array was used to semi-quantitatively analyze a panel of 105 targeted cytokines (R&D Systems, MN, United States). Based on the results of the array and previously cited literature, TNF-α, ICAM-1, HGF, SDF1-α levels were selected to be quantitatively analyzed using the corresponding ELISA immunoassay method (Human TNF-α Quantikine HS ELISA, Human ICAM-1/CD54 Allele-specific Quantikine ELISA Kit, Human HGF Quantikine ELISA Kit, Human CXCL12/SDF-1 alpha Quantikine ELISA Kit; R&D Systems, Minnesota, United States).
Quantification of VEGF and SDF-1α Release
CXCL12 Quantification from Conditioned Media
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