At 24 h post administration, mice were bled for sera and killed for tumor collection. The obtained tumor was added to tissue lysate (absin, abs9225, Shanghai, China) at 10 mg tumor tissue/50 μL tissue lysate and homogenized using a tissue homogenizer. The supernatant was collected by centrifugation. Both sera and tumor tissue lysates were collected for CCL2 cytokine detection by mouse CCL2 ELISA kits (absin, abs520016).
Abs9225
Manufactured by Absin
Sourced in China
The Abs9225 is a versatile laboratory equipment designed for a range of scientific applications. It features a compact and durable construction, with precise temperature control and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Tissue lysate, by Absin (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
P drp1, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Iseq00010, by Merck Group (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
A44114, by Thermo Fisher Scientific (1 mentions)
Abs920, by Absin (1 mentions)
Ox camkii, by GeneTex (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
5 protocols using abs9225
1
CCL2 Cytokine Detection in Tumor Tissues
2
Protein Isolation and Western Blot Analysis
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Proteins were isolated from spinal cord using RIPA (abs9225, Absin, Shanghai, China) with protease inhibitor (BP101, Bio-Platform, Shanghai, China), and the concentration was determined with BCA Protein Assay Reagent (BP104, Bio-Platform, Shanghai, China). Protein samples were mixed with sample buffer and boiled for 10 min. For western blotting, samples containing 20 ug protein were loaded, separated by electrophoresis, and then transferred onto polyvinylidene difluoride membranes (0010, Merck Millipore Ltd., MA, USA) using sandwich method. An overnight incubation at 4 °C with primary antibodies against AMPK (5831, Cell Signaling Technology, Boston, MA, USA), p-AMPK (2535, Cell Signaling Technology, Boston, MA, USA), Drp1 (8570, Cell Signaling Technology, Boston, MA, USA), p-Drp1 (3455, Cell Signaling Technology, Boston, MA, USA) was performed before incubating with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The protein bands were visualized by enhanced chemiluminescence (P10300, NCM, Suzhou, China) and photographed by an image system (5200, Tanon, Shanghai, China).
3
Western Blot Analysis of SFRP1
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The treated cells were routinely harvested and lysed by a cell lysis buffer (abs9225, Absin, China) [17 (link)]. After centrifugation, the protein concentration of the cell supernatant was examined using a BCA kit (abs9232, Absin, China). After denaturation, equal quantities of protein were electrophoresed and then transferred onto a polyvinylidene difluoride membrane (#ISEQ00010, Merck-Millipore, USA). After blocking, they were incubated with primary antibody overnight at 4℃ followed by the addition of secondary antibody (S0001, Affinity, USA) for 1 h at 37℃. Finally, protein expression was examined with a color reagent (abs920, Absin, China) in a chemic luminous instrument gel imaging system (A44114, Invitrogen, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed for the housekeeping gene. The primary antibodies of SFRP1 (1:1,000, 35 kDa, #3534) and GAPDH (1:1,000, 37 kDa, #2118) were obtained from CST (USA).
4
Comprehensive Cytokine Profiling in Murine Tumor
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A microarray containing 31 chemokines was applied to characterize the cytokine profiles in the serum and tumor tissues of mice with the Luminex multiple cytokines assay platform. Nineteen serum samples (n = 10 in the T group, n = 9 in the TD group) were diluted twofold before use. Three tumor tissues in each group were randomly selected for preliminary cytokine screening. Tissue samples were preprocessed with a lysis buffer specialized for multifactor detection (abs9225, absin, Shanghai). The concentration of the obtained total protein was adjusted to 6 μg/μL. A 96-well microplate was added to 50μL of bead mixture in each well and placed on a magnetic frame for bead adsorption. After two washes, fifty microliters of standard product and sample were added and incubated for 30 minutes with shaking at room temperature. The microplate was placed on a magnetic frame and washed three times, and then 25 μL of diluted biotin-labeled antibody complexes was added to each well and incubated for 30 minutes. Afterward, fifty microliters of diluted streptavidin-tagged PE were added for a 10-minute reaction, and the plate was washed three times. Each well was resuspended in 125 μL bead-containing assay buffer, and the plate was loaded in a Luminex 200 for detection.
5
Quantifying Protein Expression via Western Blot
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Total proteins were prepared using RIPA lysis buffer (abs9225, Absin, Shanghai, China) containing 1% protease inhibitor (BP101, Bio-Platform, Shanghai, China). Tissue lysates were separated by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010, Merck Millipore Ltd., MA, USA) by the sandwich method. The membranes were incubated with primary antibodies against CB2R (PA569179, Thermo Fisher, Shanghai, China), CaMKII (4436, Cell Signaling Technology, Boston, MA, USA), and ox-CaMKII (36254, GeneTex, Southern California, CA, USA) overnight at 4°C followed by a horseradish peroxidase peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were detected by enhanced chemiluminescence (P10300, NCM, Suzhou, China) and were scanned by a Tanon image analysis system (5200, Tanon, Shanghai, China).
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