We analyzed a separate group of three AIR eyes and five control subjects with epiretinal membranes (ERMs) and IMHs (Table 1 ; Cohort 2). Vitreous samples of this cohort were analyzed using multiplex ELISA as previously described.7 (link) Briefly, vitreous cytokine signaling proteins were measured using the Human Cytokine Quantibody Array 4000 (RayBio) per the manufacturers protocol. This array concurrently detected and processed 200 human proteins. First, the array chips were incubated with sample diluents for 30 minutes at room temperature to act as a block. Vitreous sample volumes under 500 μL were diluted with 1x lysis buffer (RayBiotech, Norcross, GA) to reach a required sample volume. Vitreous (100 μL; 4 technical replicates per sample) was then added to the wells of the array and incubated overnight at 4 °C. A standard protein dilution was added to the wells of the array to determine protein concentrations. For signal detection, 80 μL of Cy3-streptavidin was added to each well, rinsed and visualized by laser scanner. The RayBio® Analysis Tool (RayBio®, Norcross, GA) was used for protein classification. Final protein concentrations (in pg/mL) were corrected for sample dilution.
Raybio analysis tool
Manufactured by RayBiotech
Sourced in United States
The RayBio® Analysis Tool is a laboratory equipment designed for data analysis and visualization. It provides users with a comprehensive set of features to process and interpret experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Human cytokine quantibody array 4000, by RayBiotech (2 mentions)
Lysis buffer, by RayBiotech (2 mentions)
Protein assay method, by Bio-Rad (1 mentions)
Mouse cytokine array c3, by RayBiotech (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Human cytokine antibody array, by RayBiotech (1 mentions)
6 protocols using raybio analysis tool
1
Multiplex ELISA Analysis of Vitreous Cytokines
2
Cytokine Profile in Mouse Bladder
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Mice were euthanized 48 hours after intravesical instillation of PBS or BacPAK6. Bladders were harvested and weighed. Bladder homogenates were obtained by adding 1 ml of a tissue lysis buffer (Fermentas, Maryland, USA) with a protease inhibitor cocktail (Merck, Darmstadt, Germany) to 50 mg of tissue sample and homogenizing by sonication. Bladder homogenates were then centrifuged at 16,000 × g for 30 min at 4 °C and the supernatants collected. Protein concentrations of the supernatants were determined by the Biorad protein assay method (Biorad, California, USA). An aliquot of the supernatant containing 80 μg of total protein concentration was loaded onto the Mouse Cytokine Array C3 (Raybiotech, Norcross, GA) to measure expression levels of cytokines and chemokines. RayBio® Analysis Tool (Raybiotech) was used to correlate the average signal intensities to relative expression levels of cytokines.
3
Quantitative Cytokine Profiling of Undiluted Cell Media
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Two hundred microliters of undiluted medium was applied to the Human Quantibody® Human Inflammation Array 3 (RayBio, Norcross, GA) according to the manufacturer’s instructions. This array concurrently detected and processed 40 human cytokines. First, after the glass slide was completely air dried, the array chips were incubated with sample diluents for 2 h at room temperature to act as a block. Undiluted medium (100 μl) was then added to the wells of the array and incubated for 2 h at room temperature. A standard cytokine dilution was added to the wells of the array to determine protein concentrations. For signal detection, 80 μl of Cy3-streptavidin was added to each well, and the array was rinsed and visualized by laser scanner. The RayBio® Analysis Tool (RayBio®, Norcross, GA) was used for protein classification. The flow diagram for cytokine array is shown in Supplementary Fig. 9 , and raw data are listed in the Source Data file (raw data of the cytokines array).
4
Quantifying Schwann Cell Protein Levels
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Protein levels were quantified in passage three hESC- and iPSC-derived Schwann cells with the Human L Series Array 1000 Membrane Kit (RayBiotech, AAH-BLM-1000-2). following the manufacturer’s protocol. Briefly, cell lysates were biotinylated and incubated on membranes containing antibodies for 1000 human proteins. Immunoblot with HRP-conjugated Streptavidin was then performed and the membranes were imaged. Relative protein levels were quantified using the RayBio Analysis Tool and signal intensity of all samples were normalized to the negative control value for GM01582 iPSC-derived Schwann cells. Signal intensity for each protein was averaged and compared between hESC- and hiPSC-derived Schwann cells (n=1 per stem cell line, n=3 per stem cell type) and compared by multiple unpaired t-tests (without and with Welch correction for unequal variance) with the false discovery rate approach two-stage step-up method of Benjamini, Kreiger and Yekutieli method in Prism 9 (desired FDR = 1%, significant p<0.05).
5
Vitreous Cytokine Signaling in ERM and IMH
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We analyzed a separate group of 3 AIR eyes and 5 control subjects with ERMs and IMHs (Table 1 ; Cohort 2). Vitreous samples of this cohort were analyzed using multiplex ELISA as previously described.7 (link) Briefly, vitreous cytokine signaling proteins were measured using the Human Cytokine Quantibody Array 4000 (RayBio) per the manufacturer’s protocol. This array concurrently detected and processed 200 human proteins. First, the array chips were incubated with sample diluents for 30 minutes at room temperature to act as a block. Vitreous sample volumes under 500 μl were diluted with 1× lysis buffer (RayBiotech) to reach a required sample volume. Vitreous (100 μl; 4 technical replicates per sample) was then added to the wells of the array and incubated overnight at 4 °C. A standard protein dilution was added to the wells of the array to determine protein concentrations. For signal detection, 80 μl of Cy3-streptavidin was added to each well, rinsed, and visualized by laser scanner. The RayBio Analysis Tool (RayBio) was used for protein classification. Final protein concentrations (in pg/ml) were corrected for sample dilution.
6
Biotin-Labeled Cytokine Profiling of hUCB-MSCs
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Growth medium was collected from the primary neurons and co-cultures of neurons and hUCB-MSCs with or without Aβ42 peptide treatment. In order to detect the proteins secreted from the hUCB-MSCs, a biotin-label-based Human Cytokine Antibody Array (RayBiotech. Inc., Norcross, GA, USA) was conducted according to the manufacturer’s protocol. Each conditioned medium was mixed with a biotin-labelling reagent. The glass chips were incubated in blocking buffer at room temperature and in each biotin-labelled conditioned medium for 2 h at room temperature (RT). After washing, the glass chips were incubated with streptavidin-conjugated fluorescent dye for 2 h at RT. After another wash and final drying step, signals from the glass chips were detected with a VIDAR Revolution 4200 Laser Scanner (VIDAR Systems Corp., Herndon, VA, USA), and normalised signal intensity was acquired with Analysis Tool software (RAYBIO® ANALYSIS TOOL) provided by RayBiotech.
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