To determine the resting serum levels of BDNF, human serum samples were diluted 1:20 with the appropriate buffer. Human serum BDNF concentrations were measured in duplicates using enzyme-linked immunosorbent assay (ELISA) kit according to the instructions of the manufacturer (Quantikine Human Free BDNF ELISA kit, R&D systems, Minneapolis, MN). Using this assay the minimum detectable dose of human free BDNF is less than 20 pg/ml. The intra-assay coefficient of variability lies between 3.8% and 6.2% while the inter-assay precision is between 7.6 and 11.3%. BDNF levels were calculated using a standard curve supplied by the ELISA kit.
Human free bdnf quantikine elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human Free BDNF Quantikine ELISA Kit is a quantitative sandwich enzyme immunoassay designed for the measurement of free brain-derived neurotrophic factor (BDNF) levels in human cell culture supernates, serum, and plasma samples.
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13 protocols using human free bdnf quantikine elisa kit
1
Quantification of Serum BDNF Levels
2
Quantification of Plasma BDNF and Cortisol
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Before each blood sampling, the catheter was flushed with saline solution. The blood samples were collected into a heparinized and centrifuged immediately at 6000 rpm at 4 • C for 3 min to separate the plasma. Thereafter, separated plasma samples were transferred to Eppendorf tubes and stored at -80 • C until analysis. For the quantification of plasma BDNF (pBDNF) levels, samples were analyzed in duplicate using the Quantikine Human Free BDNF ELISA kit (#DBD00, R&D Systems, Inc., Minneapolis, MN, United States) according to manufacturer's instructions. For the analysis of plasma levels of cortisol, samples were analyzed in duplicate by an ELISA kit (#CO368S, Calbiotech Inc., El Cajon, CA, United States) according to the manufacturer's instructions. Plasma levels of BDNF and cortisol are expressed in pg × mL -1 and ng × mL -1 , respectively.
3
Comprehensive Assessment of Cardiometabolic Health
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All measurements were performed during a 3-week period before randomization and after 12 weeks of intervention. Weight was measured in light clothing and length on a digital height-measuring gauge. Waist circumference was measured midway between the iliac crest and lower rib during exhalation. Body composition was estimated using dual energy X-ray absorptiometry (GE Medical Systems, Lunar Prodigy X-ray Tube Housing Assembly, Brand BX-1L, Model 8743, Madison, WI, USA).
Fasting serum triglycerides, cholesterol, HDL cholesterol, insulin, HbA1c and blood glucose levels were analyzed with routine clinical laboratory methods at the Department for Clinical Chemistry, Umeå University Hospital. The homeostatic model assessment (HOMA-IR; fasting glucose × fasting insulin/22.5) was used to estimate insulin resistance. LDL cholesterol was calculated as (serum cholesterol—serum HDL—serum triglycerides)/2.2. Fasting serum BDNF levels was analyzed in duplicates with an inter- and intra-assay coefficient of variation <15% (Human Free BDNF Quantikine ELISA Kit, R&D systems, Abingdon, United Kingdom).
Aerobic capacity was estimated by using a standardized cardiopulmonary exercise test and physical activity measured with a combined heart-rate monitor and accelerometer (Actiheart®, CamNtech Ltd., Cambridge, United Kingdom) for 7 consecutive days.
Fasting serum triglycerides, cholesterol, HDL cholesterol, insulin, HbA1c and blood glucose levels were analyzed with routine clinical laboratory methods at the Department for Clinical Chemistry, Umeå University Hospital. The homeostatic model assessment (HOMA-IR; fasting glucose × fasting insulin/22.5) was used to estimate insulin resistance. LDL cholesterol was calculated as (serum cholesterol—serum HDL—serum triglycerides)/2.2. Fasting serum BDNF levels was analyzed in duplicates with an inter- and intra-assay coefficient of variation <15% (Human Free BDNF Quantikine ELISA Kit, R&D systems, Abingdon, United Kingdom).
Aerobic capacity was estimated by using a standardized cardiopulmonary exercise test and physical activity measured with a combined heart-rate monitor and accelerometer (Actiheart®, CamNtech Ltd., Cambridge, United Kingdom) for 7 consecutive days.
4
Preparation of Conditioned Media for Cell Studies
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Conditioned medium samples were obtained from HEK293T cells transfected with pVax1, pVax1-hBDNF [10 (link)], pVax1-huPA, or pNCure-fIntr [4 (link)], denoted as Control, BDNF, uPA, and BU (BDNF + uPA), respectively. For this endeavor, transfected HEK293T cells were cultured in serum-free DMEM High Glucose for 48 h. Then medium samples were removed, centrifuged for 10 min at 3000× g to remove cell debris, and concentrated ~50 times using a Centriprep Centrifugal Filter Unit (Merck, #4302, Tullagreen, Ireland) until a hBDNF concentration of 3.5 ng/µL (108 nM) was reached, confirmed using the Human Free BDNF Quantikine ELISA Kit (R&D Systems, #DBD00, Minneapolis, MN, USA). The huPA concentration in uPA and BU medium samples was adjusted to 0.4 ng/µL (8 nM), confirmed with the uPA (URK) Human ELISA Kit (Abcam, #ab119611, Waltham, MA, USA).
5
Comprehensive Cytokine and Cell Profiling
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Unless otherwise indicated, all reagents were obtained from Sigma: IMDM, penicillin, streptomycin, Trizol, unbuffered RPMI-1640 (Life Technologies); anti-fibroblast magnetic microbeads (Miltenyi Biotech); FBS, DMEM (Euroclone); glucose (Merk); MuLV Reverse Transcriptase (Applied Biosystems); RANKL, M-CSF (Peprotech); nitrocellulose membrane, D-luciferin (Thermo Fisher Scientific); BCA protein assay, Restore Western Blot Stripping buffer, Pierce ECL 2 Western Blotting Substrate (Pierce); TruSeq RNA Sample Prep Kit v2, Cycle Sequencing v4 regents (Illumina); Human IL6 DuoSet ELISA Kit, Human CXCL8/IL8 Quantikine ELISA Kit, Human Free BDNF Quantikine ELISA kit (R&D Systems Inc.). The antibodies used were: anti-rabbit Alexafluor, anti-mouse Alexafluor (Molecular Probes); Anti-MCT4 (sc-50329), anti-TBP (sc-204, Santa Cruz Biotechnology); anti-cytokeratin (M0821, Dako); anti-cytokeratin-TRITC (41-9003, Affimetrix eBioscience); anti-LAMP2 (HPA029100); anti-IL8 (AB18672, Abcam); anti-vimentin (sc-6260, Santa Cruz Biotechnology); omeprazole (Sandoz); ketamine (MSD Animal Health Srl). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit were obtained from GE Healthcare.
6
Quantifying Growth Factors in Culture
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Culture medium was collected every 2 days and stored −20 °C to measure growth factor concentrations. BMP-2 and BDNF were measured by ELISA (BMP-2 human ELISA kit, Thermo Fisher Scientific and Human Free BDNF Quantikine ELISA kit, R&D systems); a microplate reader was used to measure the absorption of each sample at 495 nm. The amount of BDNF and BMP-2 was calculated using a calibration curve based on the known concentration. This experiment was carried out in triplicate.
7
Biomarker Measurement Protocol for Plasma
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Blood extraction was performed between 8:00 and 9:00 a.m. following an overnight fast by nurses in the Primary Health Care Centers. All participants were instructed to not exercise 8 h before the blood test. Blood samples were obtained from the antecubital vein and collected in EDTA tubes for plasma analyses. Tubes were immediately transferred to the IGTP-HUGTP Biobank integrated in the Spanish National Biobanks Network of Instituto de Salud Carlos II (PT13/0010/0009) and Tumor Bank Network of Catalonia, and they were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees. Plasma aliquots were stored at −80°C.
As Level 1 outcomes, we obtained peripheral BDNF levels using an ELISA kit (Human Free BDNF Quantikine ELISA Kit; R&D Systems, Minnesota, USA). The rest of the molecular markers were selected according to the Projecte Moviment trial (Castells-Sánchez et al., 2019 (link)). TNF-α, ICAM-1, HGF, and SDF1-α levels were analyzed quantitatively using the corresponding ELISA immunoassay method (Human TNF-α Quantikine HS ELISA, Human ICAM-1/CD54 Allele-specific Quantikine ELISA Kit, Human HGF Quantikine ELISA Kit, Human CXCL12/SDF-1 alpha Quantikine ELISA Kit; R&D Systems, Minnesota, USA).
As Level 1 outcomes, we obtained peripheral BDNF levels using an ELISA kit (Human Free BDNF Quantikine ELISA Kit; R&D Systems, Minnesota, USA). The rest of the molecular markers were selected according to the Projecte Moviment trial (Castells-Sánchez et al., 2019 (link)). TNF-α, ICAM-1, HGF, and SDF1-α levels were analyzed quantitatively using the corresponding ELISA immunoassay method (Human TNF-α Quantikine HS ELISA, Human ICAM-1/CD54 Allele-specific Quantikine ELISA Kit, Human HGF Quantikine ELISA Kit, Human CXCL12/SDF-1 alpha Quantikine ELISA Kit; R&D Systems, Minnesota, USA).
8
Quantitative BDNF and Cytokine Analysis
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Enzyme-linked immunosorbent assay (ELISA) was performed to confirm expression level of BDNF. Three different passages of BDNF-eMSCs were used for ELISA assay. BDNF-eMSCs were seeded at a density of 1 × 105 cells per 12-wells (Corning) with 1 mL complete media. After 48 h in the 37 °C, 5% CO2 incubator, supernatants were harvested and centrifuged to remove cell debris. BDNF concentrations were determined using a Human Free BDNF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. The absorbance of the samples at 450 nm was measured using SpectraMAX 190 (Molecular Devices, San Jose, CA, USA) microplate reader.
BDNF levels were determined in cell culture media using a ProcartaPlex Multiplex ELISA Kit according to the manufacturer’s protocol (eBioscience, Wien, Austria). Brain inflammatory cytokines, including interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, were estimated in the peri-infarct area of the brain tissues using a MILLIPLEX MAP ELISA Kit, according to the manufacturer’s protocol (EMD Millipore, Billerica, MA, USA).
BDNF levels were determined in cell culture media using a ProcartaPlex Multiplex ELISA Kit according to the manufacturer’s protocol (eBioscience, Wien, Austria). Brain inflammatory cytokines, including interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, were estimated in the peri-infarct area of the brain tissues using a MILLIPLEX MAP ELISA Kit, according to the manufacturer’s protocol (EMD Millipore, Billerica, MA, USA).
9
BDNF Secretion Quantification
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BDNF secretion was quantified in cultures of control and BDNF-transfected cells after incubation for a defined time in a defined volume of medium. Supernatants were analyzed using the Human Free BDNF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. For quantification as a function of the cell number, cells were trypsinized with 0.05% trypsin-EDTA and counted. Counting of ARPE-19 cells was performed using the CASY Cell Counter TT (Roche, Basel, Switzerland). Primary hRPE cells were mixed with trypan blue solution and counted using a hemocytometer.
10
Quantitative BDNF and IGFBP-2 ELISA
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Serum quantitative determination of BDNF was performed by ELISA using the Human Free BDNF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. The detection range was 62.5–4000 pg/mL, and the sensitivity was 20 pg/mL. Serum insulin-like growth factor binding protein-2 (IGFBP-2) concentration was determined by ELISA. The measurement of all other analytes reported here was described in previous papers [17 (link),18 (link)].
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