THP-1 cells were differentiated as previously described in this manuscript. Following differentiation, cells were treated with 10 μM Takinib or DMSO vehicle control. 24 hours after treatment, supernatant was added to Human Cytokine XL proteome array (R&D Systems), or Angiogenesis proteome array. Apoptosis biomarkers were visualized with the Apoptosis Array kit (R&D Systems). All procedures were conducted in accordance with manufacturer protocol. Chemiluminescence was used to visualize protein quantities.
Apoptosis array kit
Manufactured by R&D Systems
Sourced in United States
The Apoptosis Array Kit is a multiplex ELISA-based assay designed to detect and quantify the levels of multiple apoptosis-related proteins simultaneously in a single sample. The kit provides a comprehensive analysis of key apoptosis regulators and effectors, enabling researchers to gain a broader understanding of apoptotic pathways and responses.
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Lab products found in correlation
Human cytokine xl proteome array, by R&D Systems (1 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Rh csf1, by R&D Systems (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Rhrankl, by R&D Systems (1 mentions)
Enhanced chemi luminescence (ecl), by Sartorius (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
5 protocols using apoptosis array kit
1
Cytokine and Angiogenesis Profiling
2
Inhibitory Effects of mAb 3.1 on Feline Osteoclasts
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To further analyze the inhibitory properties of mAb 3.1, feline osteoclasts were targeted with the antibody. Feline bone marrow cells were differentiated for 10 days in rhRANKL (30 ng/ml, R&D Systems) and rhCSF-1 (10 ng/ml, R&D Systems) in low adherence tissue culture plates (Corning) (kindly provided by Seungmee Lee (Roslin Institute)). Anti-CSF-1R mAb 3.1 hybridoma supernatant or an irrelevant anti-CCR2 hybridoma supernatant was added to the cells at 1/6 of the total culture volume. One control well received no CSF-1 after the initial differentiation of the cells. The culture medium and supplements were renewed at 48 h. Cells were lysed on day 4 using 1 ml/well of lysis buffer 15 (R&D Systems, Apoptosis Array Kit) for 30 min at 4°C. The protein mixture was assessed by western blotting. Membranes were probed with anti-pAkt (Ser 473) and total Akt (both Cell Signaling). Antibodies were used sequentially on the same membrane, after stripping the nitrocellulose membrane using Restore PLUS Western Blot Stripping Buffer (Thermo Scientific). Secondary antibody was swine anti-rabbit HRP-conjugated.
3
Apoptosis Protein Expression Profiling
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To analyze the expression profiles of apoptosis-related proteins, we used an Apoptosis Array Kit (R&D Systems) and performed assays with 35 antibodies specific for apoptosis-related proteins. These proteins were subsequently blotted on nitrocellulose membranes in duplicate. Briefly, 1*107 cells were harvested, and whole-cell lysates were extracted, according to manufacturer's instructions. Then, 400 μg of protein was mixed with 15 μl of biotinylated antibodies. After pretreatment, the samples were incubated with the assay membranes overnight at 4°C. After the membranes were washed, they were treated sequentially with streptavidin-HRP and chemiluminescent detection reagents. After 2 min, the signals on the X-ray film were quantified by a transmission-mode scanner, and the array images were analyzed using Lane 1D (Sage, China). The average background signal was eliminated, and the arrays were calibrated according to the signal strength of the positive controls. The average signal (integrated pixel density) for each apoptosis-related protein was determined, and the corresponding signals for each protein in different arrays were compared.
4
Curcumin Induces Caspase 3 Activation
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ChaGo-K-1 cells were incubated with hCG for 24 h and subsequently with curcumin (40 μM) for 24 h. Conversion of pro-caspase 3 to active (cleaved) caspase 3 was assessed using an apoptosis array kit (R&D Systems). Reactive spots were visualized by enhanced chemiluminescence (ECL; Biological Industries).
5
Profiling Apoptotic Factors in Tumor Tissues
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The apoptotic factors expressed by the tumors were collected from BALB/c nude mice and detected using cytokine chip analysis. Briefly, total protein was extracted from tumor tissue and its amount was determined using a BCA Protein Assay Kit (Merck Millipore, Billerica, MA, USA). An Apoptosis Array Kit (ARY009, R&D Systems, Millipore, USA) was used to assess the presence of 35 factors in the total protein, following protocols outlined by the manufacturer.
The tumors obtained from the BALB/c mice were used to detect 308 mouse proteins. Total protein was extracted from tumor tissue using ice-cold tissue protein extraction reagent containing 0.5% protease inhibitor cocktail, phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktail. The total protein amount was determined using a BCA Protein Assay Kit. A RayBio® L-Series Mouse Antibody Array 308 Glass Slide Kit (RayBiotech, AAM-BLG-1, USA) was used to assess the presence of 308 factors in the total protein, in accordance with the manufacturer’s protocols.
The tumors obtained from the BALB/c mice were used to detect 308 mouse proteins. Total protein was extracted from tumor tissue using ice-cold tissue protein extraction reagent containing 0.5% protease inhibitor cocktail, phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktail. The total protein amount was determined using a BCA Protein Assay Kit. A RayBio® L-Series Mouse Antibody Array 308 Glass Slide Kit (RayBiotech, AAM-BLG-1, USA) was used to assess the presence of 308 factors in the total protein, in accordance with the manufacturer’s protocols.
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