Seven (synaptophysin), 8 (GFAP), or 9 (Aβ1–42, cPARP, BDNF, IL-1β, TNFα) days after Aβ25–35 or Sc.Aβ injection, hippocampi were dissected out and rinsed in ice-cold PBS to remove excess blood, and weighed before nitrogen freezing and −80°C storage. Tissues were cut into small pieces, homogenised, sonicated and centrifuged. The supernatants were assayed immediately by ELISA for GFAP, synaptophysin, IL-1β (USCN, China), TNFα (Thermo Scientific, France), BDNF (Promega, France), cPARP (Cell Signalling, France) and Aβ1–42 (Euromedex, France) following manufacturer's recommendations. All samples were assayed in duplicate and only samples for which the CV was <25% between replicates were included for analyses. Results were expressed in pg protein per mg of brain tissue, or as % of Sc. control. Protein concentration was determined in brain homogenates with the BCA protein assay kit (Pierce Perbio Science, France).
Brain derived neurotrophic factor (bdnf)
Manufactured by Promega
Sourced in United States, Germany
BDNF is a recombinant human brain-derived neurotrophic factor. It is a protein that promotes the survival and differentiation of existing neurons, and supports the growth and differentiation of new neurons and synapses.
Automatically generated - may contain errors
Lab products found in correlation
Hs crp, by Tecan (3 mentions)
Il 1β, by Thermo Fisher Scientific (3 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Cxcl5 rantes, by R&D Systems (2 mentions)
Cxcl12 sdf 1α, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Cortisol, by Tecan (2 mentions)
Sgp130, by RayBiotech (2 mentions)
Epidermal growth factor (egf), by R&D Systems (2 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Nerve growth factor (ngf), by Promega (2 mentions)
Aβ1 42, by Fujifilm (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Multi beads shocker, by Yasui Kikai (2 mentions)
C parp, by Cell Signaling Technology (1 mentions)
Il 1β, by USCN (1 mentions)
Ldh detection kit, by Nanjing Jiancheng (1 mentions)
Caspase 3 activity kit, by Beyotime (1 mentions)
Il 10, by MultiSciences Biotech (1 mentions)
Interleukin 4 (il 4), by MultiSciences Biotech (1 mentions)
25 protocols using brain derived neurotrophic factor (bdnf)
1
Quantifying Hippocampal Markers in Alzheimer's
2
Plasma Biomarker Quantification Protocol
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To obtain plasma upon samples arriving at the lab, whole blood samples were centrifuged at 1650g for 25 min at room temperature. Subsequently, plasma samples were bio-banked at −80° C until study completion, after which all the samples were assayed in one batch. We employed commercially available ELISA kits to quantify the level of three plasma biomarkers, namely BDNF (Promega Corporation, Madison, WI, USA), hs-CRP (Tecan, Männedorf, Switzerland), and DHEA-S (CUSABIO, Houston, TX, USA). All experiments were performed as per instructions of respective manufacturers of the kits. For quantifying only the mature form of BDNF, we adhered to the protocol’s instructions in pre-processing the samples before running ELISA. Specifically, to measure total BDNF using this assay, an acid treatment to the plasma sample is typically performed. However, for the purpose of this study to only measure free mature BDNF, the acid treatment procedures were skipped. Coefficient variations (CVs) of all the assays were <10%. All personnel who processed the samples and ran the ELISA were blinded to the diagnostic status of the participants.
3
Quantifying Plasma BDNF and LDH in Rat and Human
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Plasma BDNF level from rats and human were measured by sandwich ELISAs according to the manufacturer's instructions for BDNF (Promega, USA). Assays were performed on polystyrene 96-well plates. The BDNF concentration was quantified against a standard curve calibrated with known amounts of protein. The BDNF ELISA systems have low cross-reactivity (<3%) with other related neurotrophic factors. Each value is a mean of duplicated measurement. Serum LDH activity was measured using LDH detection kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instruction. Caspase-3 activity was measured using caspase-3 activity kit (Beyotime, China) according to the manufacturer's protocol.
4
Measurement of Neuroinflammatory Markers
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The mentioned culture supernatants yielded here were collected to cytokine concentrations of IL-1β, IL-4, IL-6, IL-10, TNF (Multisciences, China), and the secretion of neurotrophic factor BDNF (Promega, USA) in the brain by a sandwich ELISA kits in line with the instructions of the manufacturer. The determinations were conducted in duplicate. The results are denoted as pg/mL.
5
Neuroprotective Effects of CPE-ΔN in Neurons
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Rat hippocampal or cortical neurons were treated with 1 µM dexamethasone (Sigma) for 24 h. The cells were then harvested, extracted and analyzed for expression of CPE-ΔN. FGF2 (R&D) or BDNF (Promega) was added to neurons for 15 min. The cells were then harvested, extracted and analyzed for activation of ERK and AKT. In other experiments, primary neurons were transduced with adenoviral vectors (Type 5 (dE1/E3)), carrying the cDNA of CPE ΔN (custom made by Vector Biolabs) or LacZ (Vector Biolabs) as a negative control, at 50 MOI for 72 h. The neurons were then treated with 100 µM H2O2 (Sigma), 40 µM glutamate (Sigma) or 0.4 µM staurosporine (Sigma) for 24 h. In specific experiments, 10 µM LY294002 (Cell Signaling), 5 µM U0126 (Cell Signaling), 1 µM PD166285 (Sigma) or SU5402 (Sigma) were added to the neurons during the 20–24 h treatment with glutamate, staurosporine or H2O2. Cell viability, cell cytotoxicity and TUNEL assays were then used to measure cell death after the treatments.
6
Plasma Biomarkers Measurement Protocol
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To measure the biomarkers, ten millilitres of fasting venous blood were withdrawn from each participant between 8.30 a.m. and 9.30 a.m. at the research centre. The blood was sent to the laboratory within three hours after the first venepuncture. Subsequently, the whole blood samples were centrifuged at 1650 g for 25 min at room temperature to obtain the plasma. All the blood and plasma samples from both groups at the three time-points were stored at −80 °C. The nine plasma biomarkers were examined using commercially available enzyme-linked immunosorbent assay (ELISA) kits, namely, IL-6, IL-1β (ThermoFisher, Waltham, MA, USA), CXCL12/SDF-1α, CXCL5/RANTES (R&D Systems Inc., Minneapolis, MN, USA), BDNF (Promega Corporation, Madison, WI, USA), DHEA (CUSABIO, Houston, TX, USA), cortisol, hs-CRP (Tecan, Männedorf, Switzerland), and sgp130 (RayBiotech Inc., Norcross, GA, USA). Assays for each biomarker were run on the same day to avoid batch effect.
7
Plasma Biomarker Profiling Protocol
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To obtain plasma, whole blood samples were centrifuged at 1650g for 25 minutes at room temperature. Subsequently, plasma samples from the 3 time points were bio-banked at −80 °C until study completion. Assays for each biomarker were run on the same day after study completion to avoid batch effect. We employed commercially available enzyme-linked immunosorbent assay (ELISA) kits to measure the level of 9 plasma biomarkers, namely IL-6, IL-1β (ThermoFisher, Waltham, MA), CXCL12/SDF-1α, CXCL5/RANTES (R&D Systems Inc., Minneapolis, MN), BDNF (Promega Corporation, Madison, WI), DHEA (CUSABIO, Houston, TX), cortisol, hs-CRP (Tecan, Männedorf, Switzerland), and sgp130 (RayBiotech Inc., Norcross, GA).
8
Measurement of Neurotrophic Factors in MCAO
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The concentration of brain-derived neurotrophic factor (BDNF; Promega, Germany), glial cell line-derived neurotrophic factor (GDNF, Promega), vascular endothelial growth factor (VEGF; R&D Systems, United States), nerve growth factor (NGF; Promega), basic fibroblast growth factor (bFGF; R&D Systems) and epidermal growth factor (EGF; R&D Systems) was measured by enzyme linked immunosorbent assay (ELISA) in left (ischemic) hemispheres 84 days after MCAO as described before (Doeppner et al., 2012a (link)).
9
Fermented Dairy Effects on Alzheimer's
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To evaluate the effects of fermented dairy products on Alzheimer’s disease, 3-month-old transgenic and wild-type female mice were fed a diet with or without fermented sample (2% w/w) for three months (n = 11 mice in each group). At the age of 6 months, the mice were euthanized and the brains were removed. The left hemisphere of the brain was homogenated in RIPA buffer (Wako) with a multi-beads shocker (Yasui Kikai, Osaka, Japan). After centrifugation at 500,000 × g for 20 min, the total protein concentration of the supernatant was measured with a BCA protein assay kit (ThermoScientific, Yokohama, Japan). To quantify Aβ1–42 (Wako), MIP-1α (R&D systems, MN, USA), TNF-α (eBiosciences, CA, USA), IL-1β (eBiosciences), synaptophysin (Life Science Inc., FL, USA), BDNF (Promega, WI, USA), GDNF (Promega), and NGF (Promega) in the hippocampus, an appropriate ELISA kit was used. The right hemispheres were fixed in 10% formalin (Wako) and analyzed immunohistochemically using the following specific antibodies: anti-Aβ1–42 (polyclonal, Invitrogen, CA, USA), anti-Iba-1 (polyclonal, Wako), and anti-MIP-α (polyclonal, R&D systems).
10
Luciferase Assay for miRNA Function Assessment
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Rat hippocampal (DIV5 to DIV11) and cortical neurons (DIV4 to DIV6) from three independent biological replicates were transfected respectively in technical triplicates with 200 ng or duplicates with 100 ng of Firefly and Renilla luciferase constructs. At the indicated DIV after transfection, cells were pre-incubated in APV as described before, further stimulated with BDNF for 6 h and lysed in passive lysis buffer (Promega). For pmirGLO reporter transfections, DIV5 rat cortical cells from four independent biological replicates were transfected with 500 ng pmirGLO constructs and with 500 ng pri-miR constructs in technical duplicates and 48 h later cells were lysed.
The activity of Firefly and Renilla luciferases was measured with a GloMax R96 Microplate Luminometer (Promega). Averages of Firefly/Renilla activity ratios from miR-134 reporters and mutants were further normalized with average ratios obtained from the activity of empty luciferase vectors, subjected to the same drug treatment/transfection conditions.
The activity of Firefly and Renilla luciferases was measured with a GloMax R96 Microplate Luminometer (Promega). Averages of Firefly/Renilla activity ratios from miR-134 reporters and mutants were further normalized with average ratios obtained from the activity of empty luciferase vectors, subjected to the same drug treatment/transfection conditions.
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