The proteins were analyzed for chemokine expression based on the instructions provided by the commercially available Mouse Chemokine Array kit (Proteome profiler Array) from R&D systems. Briefly, the proteins were isolated from different time points following treatment of the mice with NSCs. As a control, tissue from healthy mouse brain was used for the study. Protein concentration used for the study was calculated to be about 200 µg per membrane, calculated using the BCA Protein Assay kit. The experiment was performed in 4 well multidishes. Initially, the membranes containing the antibodies were blocked with blocking buffer for 1 h at room temperature. Concurrently, the detection antibody cocktail was reconstituted in buffer and added to the protein samples, incubating for 1 h at room temperature. Following the incubation, the membranes were washed 3 times with wash buffer and incubated with the secondary antibody (Streptavidin-HRP) for 30 min at room temperature. After the incubation time was complete, the secondary antibody was drained off and the membranes were washed again 3 times. The membranes are covered with the Supersignal West Pico Chemiluminescent substrate (Pierce) for detection and exposed to X-ray films for multiple exposure times. The membranes are compared for relative chemokine expression.
Mouse chemokine array kit
Manufactured by R&D Systems
Sourced in United States
The Mouse Chemokine Array Kit is a sandwich-based antibody array that allows for the simultaneous detection and quantification of multiple mouse chemokines in a single sample. The kit includes a pre-coated membrane and detection antibodies, providing a convenient platform for the analysis of mouse chemokine profiles.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (1 mentions)
Cytometric bead array cba, by BD (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Mouse cytokine array kit, by R&D Systems (1 mentions)
4 μm pore pet track etched membranes, by Corning (1 mentions)
Mouse angiogenesis array kit, by R&D Systems (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ripa buffer, by Abcam (1 mentions)
5 protocols using mouse chemokine array kit
1
Chemokine Expression Analysis in Mouse Brain
2
Comprehensive Tumor Chemokine Profiling
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Total protein content of tumor lysates was quantified by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Profiling of intratumoral chemokines was done for 500μg protein from tumor lysates using the Mouse Chemokine Array Kit (R&D Systems) according to the manufacturer’s instructions. Signal was revealed by SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific); and signal of individual chemokine spots was quantified using ImageJ software. Quantification of chemokines in tumor lysates or from cell culture supernatants was done by flow cytometry using the Cytometric Bead Array (CBA, BD Biosciences). Detection of XCL1 protein in cell culture supernatants was done by ELISA (R&D Systems). Note that the lack of sensitivity of this ELISA precluded analysis of XCL1 protein in tumor extracts.
3
Cytokine and Chemokine Profiling in Gastric Cancer
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TDMs (3 × 10⁵) were indirectly co-cultured with GCIY (1 × 10⁵) for 72 hours using transwell 6-well plates with 4-μm pore PET track-etched membranes (Corning), and then the cell culture supernatant mixture was collected by centrifugation. As a control, the supernatant of the TDM mono-culture (3 × 10⁵) was used. The assay was performed according to the manufacturer’s protocol (Human Cytokine/Chemokine Array, catalog # ARY005B, #ARY017, R&D Systems).
To investigate the humoral factors in the peritoneal cavity, the peritoneal washes were obtained from mice with or without peritoneal dissemination of GC, as described above. The washes were analyzed with Mouse Cytokine Array Kit (R&D Systems, #ARY006), Mouse Chemokine Array Kit (R&D Systems, #ARY020), and Mouse Angiogenesis Array Kit (R&D Systems, #ARY015).
To investigate the humoral factors in the peritoneal cavity, the peritoneal washes were obtained from mice with or without peritoneal dissemination of GC, as described above. The washes were analyzed with Mouse Cytokine Array Kit (R&D Systems, #ARY006), Mouse Chemokine Array Kit (R&D Systems, #ARY020), and Mouse Angiogenesis Array Kit (R&D Systems, #ARY015).
4
Chemokine Profiling in Cell Supernatants
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Sh control or sh C/EBPβ cells were seeded on a 24-well plate with 70% confluency. Their medium was changed soon after they adhered and cells were then cultured for 2 days in previously described conditions. Five hundred microliters of cell supernates were collected and centrifuged at 1500 r.p.m. for 5 min. Subsequently, pellets were discarded and supernates were used for chemokine analysis with Mouse Chemokine Array Kit (R&D Systems, Minneapolis, MN, USA; cat. no. ARY020), according to the manufacturer's protocol.
5
Quantifying Chemokine Levels in Neuroinflammation
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For the in vivo evaluation of chemokine production, the brains and spinal cords were harvested from different groups of mice on day 14 after immunization and intracardially perfused with PBS. Blood samples were harvested from mice by cardiac puncture and centrifuged at 12,800 ×g for 10 min to collect serum. Proteins were isolated by homogenizing tissues in RIPA buffer (Abcam, Cambridge, UK). Chemokine production was assessed using a Mouse Chemokine Array Kit (R&D Systems, MN, USA) according to the manufacturer's instructions. Array images were analyzed by densitometry using Image Lab Software (Bio-Rad). The data are presented as fold changes in chemokine expression compared to the corresponding positive control spots.
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