Conditioned medium from tissue culture experiments was assessed for TNF-α, IL-6 and IL-8 concentrations using commercial ELISA according to the manufacturer's instructions (Life Technologies, Mulgrave, Victoria, Australia). The concentration of mature secreted IL-1β in the media was performed by sandwich ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN USA). The concentration of PGE2 and PGF2α into the incubation media were assayed using commercially available competitive enzyme immunoassay kits according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, Waterloo, NSW, Australia). The calculated interassay and intraassay coefficients of variation (CV) were all less than 10%. Data was corrected for total protein and expressed as either ng or pg per mg protein. The protein content of tissue homogenates was determined using BCA protein assay, using BSA as a reference standard, as previously described [43] (link). For the preterm explant studies, due to patient variability, data were normalised to the untreated samples (basal), which was set at 1.
Kookaburra kits
Manufactured by Sapphire Bioscience
Sourced in Australia
Kookaburra Kits are a range of laboratory equipment designed for various scientific applications. The kits include essential tools and accessories required for conducting experiments and research. Each kit is tailored to meet specific needs, providing a comprehensive and organized solution for laboratory work.
Automatically generated - may contain errors
7 protocols using kookaburra kits
1
Secreted Cytokine Measurement in Tissue Culture
2
Cytokine and Chemokine Release Measurement
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Assessment of cytokine and chemokine release of IL6 and CXCL8 was performed using the CytoSet sandwich ELISA according to the manufacturer's instructions (Life Technologies). The release of CCL2, sICAM1, and sVCAM1 was performed by sandwich ELISA from R&D Systems according to the manufacturer's instructions. The release of PGF2α into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Kookaburra kits; Sapphire Bioscience). The inter- and intraassay coefficients of variation for all assays were less than 10%.
3
Cytokine and Prostanoid Quantification
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Conditioned medium from cell and tissue culture experiments was assessed for IL-6 and IL-8 concentrations using commercial ELISA according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). The concentration of PGE2 and PGF2α into the incubation medium were assayed using commercially available competitive enzyme immunoassay kits according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, Redfern, Australia). The calculated interassay and intraassay coefficients of variation (CV) were all less than 10%. Data was corrected for total protein and expressed as either ng or pg per mg protein. The protein content of tissue homogenates was determined using BCA protein assay (Pierce, Rockford, USA), using BSA as a reference standard, as previously described [56] (link). For the preterm explant studies, due to patient variability, data were normalised to the untreated samples (basal), which was set at 1.
4
Cytokine and Prostaglandin Release Assay
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The release of IL6 and IL8 was performed using CytoSet sandwich ELISA according to the manufacturer's instructions (Life Technologies). The limit of detection of the IL6 and IL8 assays was 16 and 12 pg/ml respectively. The release of PGE 2 and PGF 2a into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, Waterloo, NSW, Australia). The limit of detection of the PGE 2 and PGF 2a assays was 16 and 60 pg/ml respectively. For all assays, the interassay and intraassay coefficients of variation were !10%.
5
Quantification of Inflammatory Mediators
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The release of MCP-1, IL-6 and IL-8 was performed by sandwich ELISA according to the manufacturer's instructions (Life Technologies; Mulgrave, Vic, Australia). The release of PGE 2 into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience; Waterloo, NSW, Australia). The limit of detection of the PGE 2 assay was 16 pg/ml. All data were corrected for total protein and expressed as either pg or ng per mg protein. The protein content of tissue homogenates was determined using BCA protein assay (Thermo Fisher Scientific; Scoresby, Vic, Australia), using BSA as a reference standard, as previously described [18] . The calculated interassay and intraassay coefficients of variation (CV) were all less than 10%.
6
Cytokine, Chemokine, and Adhesion Molecule Release Assays
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Assessment of cytokine and chemokine release of IL6 and CXCL8was performed using the CytoSet™ sandwich ELISA according to the manufacturer's instructions (Life Technologies;
Mulgrave, Vic, Australia). The release of sICAM1 and sVCAM1 was performed by sandwich ELISA from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's instructions.
The release of PGF 2α into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, NSW, Australia). The interassay and intraassay coefficients of variation for all assays were less than 10%.
Mulgrave, Vic, Australia). The release of sICAM1 and sVCAM1 was performed by sandwich ELISA from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's instructions.
The release of PGF 2α into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, NSW, Australia). The interassay and intraassay coefficients of variation for all assays were less than 10%.
7
Cytokine and Prostaglandin Release Assays
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The release of IL6, CXCL8 and chemokine (C-C motif) ligand 2 (CCL2) was performed using CytoSet sandwich ELISA, according to the manufacturer's instructions (Life Technologies). The limit of detection of the IL6, CXCL8 and CCL2 assays was 16, 12 and 15 pg/ml respectively. The release of PGF 2a into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit, according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, Redfern, NSW, Australia). The limit of detection of the PGF 2a assay was 60 pg/ml. For all assays, the inter-and intra-assay coefficients of variation were !10%.
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