Dimension rxl autoanalyser

Extracellular acidification rate (ECAR) and oxygen consumption rates (OCR) were measured using a XF24 3 Extracellular Flux Analyzer (Seahorse). 0.5 x 10⁶ CD8⁺ T cells per well were plated on poly-D-lysine coated plates, in XF media (non-buffered RPMI base medium, 25mM glucose, 2mM glutamine and 1 mM sodium pyruvate). Assays were performed with a Seahorse Cell Energy Phenotype Test Kit, and ECAR and OCR were measured simultaneously before and after injection of Olygomycin (1 μM) and FCCP (1 μM). For experiments performed under 1% O_2, the XF24 3 analyzer was placed inside a hypoxic globe box (Coy).
Glucose and lactate levels were determined with a Dade-Behring Dimension RXL autoanalyser (Siemens). VEGF-A levels in media were measured with a VEGF-A immunoassay kit (MSD). Protein levels were normalized to viable cell counts performed on an ADAM-MC automated cell counter (NanoEnTek). T cell proliferation was measured by CFSE dilution assay 72 hours after T cell activation with plate-bound αCD3 (5 μg/ml, Biolegend) and soluble αCD28 (1μg/ml, Biolegend).

Glucose was measured by Hexokinase method on a Siemens Dimension RXL AutoAnalyser. Reagents and Calibrators were purchased from Siemens. Free fatty acids were measured using Roche Free Fatty Acid Kit. This assay was modified to run in a microtitre plate format. Thyroid-stimulating hormone (TSH), free thyroxin (fT4) and iodothyronin (T3) were measured by time-resolved fluorescence immunoassay on an AutoDELFIA analyser (Perkin Elmer) using kits from Perkin Elmer. Cortisol level was measured by fluorescence immunoassay on the Siemens Centaur Autoanalyser. A minimum of two quality control samples were run in each assay.

Umbilical plasma insulin concentration was determined using an ELISA kit (Mercodia, Uppsala, Sweden); the intra-assay coefficient of variation was 9%, and the minimum level of detection was 0.025 ng/mL. Plasma cortisol and leptin concentrations were measured by RIA as previously described (Robinson et al. 1983 (link), Blache et al. 2000 (link)). The intra-assay coefficients of variation were 11 and 5%, and the minimum levels of detection were 1.5 and 0.09 ng/mL, respectively. Plasma triiodothyronine (T3) and thyroxine (T4) concentrations were determined by RIA kits (MP Biomedicals, Loughborough, UK); the intra-assay coefficients of variation were 3 and 5%, and the minimum levels of detection were 0.14 and 7.0 ng/mL, respectively. Plasma glucose concentration was measured using a Yellow Springs glucose analyser (2300 Statplus, YSI Incorporated, Yellow Springs, Ohio, USA).
Plasma levels of total osteocalcin and the degradation products of C-terminal telopeptides of type I collagen (CTX) were determined by ELISA (Immunodiagnostics Systems Ltd, Boldon, UK). The lower limits of detection of osteocalcin and CTX were 0.5 and 0.02 ng/mL, respectively, and all measurements were made in a single assay. Total plasma calcium was measured by a Siemens Dimension RXL auto-analyser using Siemens reagents and calibrators (Siemens Healthcare, Camberley, UK).