The largest database of trusted experimental protocols

8 protocols using human cxcl12 sdf 1 alpha quantikine elisa kit

1

SDF-1α Quantification and Binding Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
SDF-1α was measured in CM from MCF-7 and MDA-MB-231 cells using a commercially available ELISA Kit in accordance with the instructions by the manufacturer (Human CXCL12/SDF-1 alpha Quantikine ELISA Kit, R&D Systems, Inc. Minneapolis, USA).
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.
+ Open protocol
+ Expand
2

Cytokine and Chemokine Profiling of MSPCs and JMML Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
BM plasma collected from one femur and one tibia in 500 µl PBS. Culture medium was collected from mouse MSPCs (4 × 106 cells per 2.0 ml) at second or third passages cultured in serum-free DMEM for 48 h. These samples were assayed for levels of IL-1β and CCL3 using enzyme-linked immunosorbent assay (ELISA) kits (IL-1β: eBioscience; CCL3: R&D Systems) following the instructions provided by the manufacturers. To determine multiple cytokines/chemokines produced by human MSPCs, MSPCs (2 × 104 cells ml−1) were cultured in serum-free StemSpan medium for 72–96 h. To determine multiple protein factors produced by cells from patients with JMML, JMML cells (2 × 105 cells ml−1) were cultured in StemSpan medium supplemented with human SCF (50 ng ml−1), human Flt3 ligand (50 ng ml−1), and human TPO (50 ng ml−1) for 72 h. The culture medium was then collected and cytokine/chemokine levels were determined by the BD Cytometric Bead Array Flex Sets (BD Biosciences) following the manufacturer’s instructions. Human CXCL12 levels in MSPC culture medium were measured using a Human CXCL12/SDF-1 alpha Quantikine ELISA Kit (R&D systems).
+ Open protocol
+ Expand
3

Quantification of Plasma Biomarkers

Check if the same lab product or an alternative is used in the 5 most similar protocols
ELISA kits were used to measure plasma levels of (C-term) SDF-1α (Human CXCL12/SDF-1 alpha Quantikine ELISA Kit by R&D system), IL-6 (Human IL-6 ELISA Pro Kit by Mabtech, Nacka Strand, Sweden) and TNF-α (TNF alpha Ultrasensitive ELISA Kit, Thermo Fisher Scientific, Waltham, MA U.S.A.) following manufacturer’s instruction. Intra- and inter-assay coefficients of variation were 3.5 and 10.6% for SDF-1α, 2.5 and 3.8% for IL-6 and 6.0 and 8.5% for TNF-α, respectively. Cytokine/chemokine concentrations were calculated in duplicate by using a standard curve generated by serially diluting reconstituted standards and by measuring the absorbance at 450 nm in a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific).
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.
+ Open protocol
+ Expand
4

CXCL12 Secretion Quantification in Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols
To confirm the upregulation of secretion of CXCL12 in CKO clones of HEK293T and UE7T-9 cells, an enzyme-linked immunosorbent assay (ELISA) was conducted. HEK293T or UE7T-9 cells of C9orf89-CKO, MAGI2-CKO, MLPH-CKO, and RHBDD2-CKO, and Non-target were seeded in 350 μL of DMEM in a 24-well plate (HEK293T: 4 × 105 cells/well, UE7T-9: 2 × 105 cells/well). After 48 h of incubation, each cell culture medium was collected and analyzed. The secreted CXCL12 level was assessed using a Human CXCL12/SDF-1 Alpha Quantikine ELISA Kit (R&D Systems), following the manufacturer’s protocol.
+ Open protocol
+ Expand
5

Biomarker Measurement Protocol for Plasma

Check if the same lab product or an alternative is used in the 5 most similar protocols
Blood extraction was performed between 8:00 and 9:00 a.m. following an overnight fast by nurses in the Primary Health Care Centers. All participants were instructed to not exercise 8 h before the blood test. Blood samples were obtained from the antecubital vein and collected in EDTA tubes for plasma analyses. Tubes were immediately transferred to the IGTP-HUGTP Biobank integrated in the Spanish National Biobanks Network of Instituto de Salud Carlos II (PT13/0010/0009) and Tumor Bank Network of Catalonia, and they were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees. Plasma aliquots were stored at −80°C.
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).
+ Open protocol
+ Expand
6

Quantifying Plasma Biomarkers in Healthy Adults

Check if the same lab product or an alternative is used in the 5 most similar protocols
Nurses in the Primary Health Care Centers obtained blood samples from the antecubital vein in EDTA tubes for plasma analyses between 8:00 and 9:00 a.m. All participants were instructed to fast overnight and not exercise 8 h before the blood test. Tubes were immediately transferred to the IGTP-HUGTP Biobank integrated in the Spanish National Biobanks Network of Instituto de Salud Carlos II (PT13/0010/0009) and Tumor Bank Network of Catalonia. They were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees and plasma aliquots were stored at −80°C.
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).
+ Open protocol
+ Expand
7

Quantification of VEGF and SDF-1α Release

Check if the same lab product or an alternative is used in the 5 most similar protocols
To determine the release of VEGF and SDF-1α, the medium was collected from the scaffolds after 1, 3, and 7 days, snap frozen with liquid nitrogen, and stored at −80 °C until analysis. The total concentrations released were determined using a Human VEGF Quantikine ELISA kit and Human CXCL12/SDF-1 alpha Quantikine ELISA kit (R&D Systems), respectively, according to manufacturer’s instructions. Due to the high sensitivity of the assay, only 5 μL of the conditioned medium was used in the VEGF analysis.
+ Open protocol
+ Expand
8

CXCL12 Quantification from Conditioned Media

Check if the same lab product or an alternative is used in the 5 most similar protocols
Conditioned media was collected and subjected to CXCL12 ELISA (Human CXCL12/SDF-1 alpha Quantikine ELISA Kit; R&D Systems, Minneapolis, MN) as directed by the manufacturer.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!