Picokine elisa kit

Peripheral blood samples from newly diagnosed patients with glioblastoma (n = 56) were collected and allowed to clot for 30 min at room temperature before centrifugation for 15 min at 1,000g. The serum was stored at −80 °C until analysis. The TSP-1 level was determined using the Quantikine immunosorbent assay kits according to the manufacturer’s instructions (R&D Systems). To confirm the functional protein-level knockdown of THBS1, the TSP-1 level was also measured in cell culture supernatants collected from scramble and THBS1-shRNA transduced HFC cells (collected one-week after infection) using the Picokine ELISA kit (Boster Biological Technology).

Transfection: Whole inner ears were dissected from C57Bl/6 mice and utricles were removed. The utricles were placed on a 0.45-µm Millicell-HA culture insert (cat# PIHA01250) in a 24-well plate with media (DMEM, N1 supplement, penicillin, neomycin 10⁻³). This process was repeated for each subsequent dissection. The tissues were incubated for 48 h at 37°C/5% CO₂ to induce injury. The vector was added to fresh media (1 µl vector/400 µl medium) into a new 24-well plate and returned to the incubator. The medium was collected and replaced every 72 h ending on the 27th day of collection. The media were then placed in −20°C freezer until further analysis. Analysis was performed in triplicate with five utricles per membrane.
Enzyme-linked immunosorbent assay (ELISA): ELISAs specific for BDNF and NT-3 were used to detect expression levels in the supernatants. Explant supernatant was thawed, and reactivity was tested for the neurotrophins (BDNF: PicoKine^™ ELISA Kit, Boster Biological Technology #EK0307/9; NT-3: mouse Neurotrophin-3 ELISA Kit, Abcam, ab213882).
A subset of neomycin medium collected from neomycin-treated explants which were incubated with Ad28.lat.bdnf were microdialyzed using Pall Nanosep^® Filters (Pall Corp, Westborough, MA United States) per manufacturer’s instructions and subsequently assayed for BDNF content as outlined above.

Specific ELISAs for mTNFα, mIL6 (Thermo Scientific, Catalog # EMTNFA and # EM2IL6, respectively), rIL6 (PicoKine ELISA Kit, Boster Biological Technology, Catalog # EK0412), and high sensitivity enzyme immunoassay (EIA) for PGE₂ (Assay Designs; Catalog # 931-001) were used to assay cytokines and PGE₂ levels in CMs from transgenic mNPsc and microglia, following the manufacturer’s instructions. The ranges of determination are: 50–2450 pg/ml for mTNFα, 7.8–500 pg/ml for mIL6; 62.5–4000 pg/ml for rIL6; 7.8–1000 pg/ml for PGE₂.
The production of nitric oxide (NO) was determined by measuring the content of nitrite, one of the end products of NO oxidation in the media, as previously described^{75 (link)}. All chemical for the NO assay were from Sigma.

In each of the SO, OVX/D-Gal, Donepezil, and E. bonariensis 100 mg/kg groups, the hippocampi of 6 rats were homogenized in ice-cold phosphate-buffered saline. Rat-specific ELISA kits acquired from My BioSource (San Diego, CA, USA) were utilized to assess the hippocampal content of Aβ42 (Cat. #MBS726579), Cytc (Cat. #MBS727663), NF-κBp65 (Cat. #MBS775083), and IL-1β (Cat. #MBS825017). Additionally, BCL-2 and BAX hippocampal contents were measured using rat ELISA kits provided by Biomatik (Ontario, Canada, Cat. # EKC40527 and EKC41377, respectively). Further, AChE was determined using an ELISA kit supplied by CUSABIO Technology LLC, China (Cat. # CSB-E11304r) and TNF-α quantification was performed using the PicoKine ELISA kit (Boster, CA, USA, Cat. #MBS175904). All procedures were performed according to the manufacturer’s guidelines. The protein content of tissue homogenates was determined as previously described (Bradford 1976 (link)).

MDCK cells were treated as stated above. Untreated MDCKs were considered as the negative control. The cell-free supernatants were harvested following 48 h incubation and stored at − 80 °C for the cytokine analysis. All the samples were tested in duplicate. The expression level of TNF-α and IL-27 following treatments was evaluated by quantitative sandwich Picokine ELISA kits (Boster Biological Technology, CA, USA) according to the manufacturer’s instructions. The optical density of the wells was measured using microplate reader (Anthos 2020, version 2.0.5) at 450 nm wavelength. The density of yellow color is proportional to the cytokine amount in the sample. The concentrations of the cytokines were calculated according to the corresponding reaction standard formula.