Cytokine and chemokine expression was measured using the Murine Proteome Profiler Mouse XL Cytokine Array (R&D Systems, Minneapolis, MN). To obtain samples, C57BL/6 mice were i. p. inoculated with either cps1-1 or cps1-1:Δgra24 (106 tachyzoites per mouse). Four days post infection the peritoneal exudate cells were collected by filling the peritoneal cavity with sterile PBS then using a 21 Ga needle to collect the cell suspension. The PECs were plated in cDMEM at 2 x 106 cells per well and were incubated for 72 hr. Following incubation, the supernatants were collected and the Murine Proteome Profiler Mouse XL Cytokine Array was used according to the manufacturer’s directions. In brief, nitrocellulose membranes were blocked for 1 hr then sample supernatants were added to the membranes. After overnight incubation, membranes were washed and a detection antibody cocktail (R&D Systems) was added. The membranes were incubated with the detection antibody for 1 hr, washed, and streptavidin-horseradish peroxidase was added. After 30 min incubation, membranes were washed three times (10 min per wash) and spots visualized by addition of enhanced chemiluminescence reagent. The membranes were imaged on a Chemi Touch Imaging System (Bio-Rad), and semi-quantitative analysis was accomplished using ImageJ software.
Detection antibody cocktail
Manufactured by R&D Systems
The Detection Antibody Cocktail is a reagent designed to facilitate the detection of target analytes in immunoassays. It contains a mixture of antibodies that are specific to the analytes of interest. The core function of the cocktail is to bind to the target analytes, enabling their identification and quantification in the assay.
Automatically generated - may contain errors
Lab products found in correlation
Chemi touch imaging system, by Bio-Rad (1 mentions)
Streptavidin hrp antibody, by R&D Systems (1 mentions)
Las4000, by GE Healthcare (1 mentions)
Proteome profiler mouse cytokine array panel a, by R&D Systems (1 mentions)
Human xl cytokine array kit, by R&D Systems (1 mentions)
Proteome profiler human xl oncology array ary026, by R&D Systems (1 mentions)
Proteome profiler human apoptosis array kit, by R&D Systems (1 mentions)
Streptavidin horseradish peroxidase, by R&D Systems (1 mentions)
5 protocols using detection antibody cocktail
1
Profiling Cytokine and Chemokine Expression in Murine Toxoplasma Infection
2
Mouse Cytokine Array Profiling
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Proteome profiler-mouse cytokine array panel A (R&D Systems) was subjected to analysis. All protocols were performed according to the manufacturer's instructions. Briefly, 1 ml of freshly collected T cell conditional medium was applied to pre-blocked cytokine array membrane. After 1 h incubation, detection antibody cocktail (R&D systems) was added and incubated with the membrane overnight at 4 °C. Streptavidin-HRP antibody (R&D systems) was used as the secondary antibody. The signals were detected by Las4000 X-ray device (GE Healthcare) and the pixel density was analyzed by Las4000 software (GE Healthcare).
3
Cytokine Profiling in Conditioned Media
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Cytokine profiling was performed using the proteome profiler Human XL cytokine array kit (# ARY022) provided by R&D Systems. Cells were incubated in serum-free media for 3 days and the conditioned media were prepared as mentioned above. The media (1 mL per membrane) were applied to the nitrocellulose-based array membrane and incubated at 4 °C overnight. Array membranes were treated with the detection antibody cocktail (R&D Systems) for 1 h and further with the streptavidin-HRP solution for 30 min. The immune complexes were visualized using the Chemi-Reagent Mix kit and the arrays were exposed to X-ray films. Based on the mean intensity of reference spots (A1/2, A23/24, and J1/2), the intensity of each dot was normalized.
4
Cytokine Profiling Using Human Cytokine Array
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A Human Cytokine Array Kit (ARY026) was purchased from R&D (Minneapolis, USA). Briefly, the conditioned medium was added into Array Buffers (R&D, ARY026) to a final volume of 1.5 mL and then applied to each antibody-printed nitrocellulose membrane for overnight incubation at 4 °C. On the next day, the membrane was incubated with the detection antibody cocktail (R&D, ARY026) for 1 h at room temperature. Treated with HRP-conjugated streptavidin antibody, the membrane was exposed to ChemiDoc. Each pair of positive dots represented signals of highly expressed cytokines and the intensity was quantified by ImageJ software. The full list of all proteins candidates is available at the manufacturer’s official website (Proteome Profiler Human XL Oncology Array ARY026: R&D Systems (rndsystems.com). Data extraction and analysis were performed after subtraction of the background and normalization to the internal references provided by the manufacturer, using an ImageJ protein array analyzer software (http://rsb.info.nih.gov/ij/macros/toolsets/Dot%20Blot%20Analyzer.txt ).
5
Apoptosis Profiling of Impaired Spermatogenesis
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The Proteome Profiler Human Apoptosis Array Kit (R&D Systems) was performed according to the manufacturer's instructions. Because of sample limitations, three samples from each group were pooled for isolation of protein. Briefly, equal amounts of proteins (250 mg) from normal spermatogenesis tissue (control) and impaired spermatogenesis biopsies (SCOS, MA, and HS) were diluted and incubated with the apoptosis array overnight at 4 C. After washing thrice, Detection Antibody Cocktail (diluted 1:100; R&D systems) was incubated for 1 hour, followed by washing thrice and incubation of streptavidin-horseradish peroxidase (diluted 1:2,000; R&D systems) for 30 minutes. After another washing step, Chemi Reagent Mix was added, and the signals on the membranes were developed. The pixel volume of the spot signals was quantified by ImageJ and normalized to reference spots.
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