Culture supernatant was collected from each growth condition, and keratinocyte growth factor (KGF-7) concentration was determined using the Quantikine human KGF/FGF-7 Immunoassay (R&D Systems Inc., Minneapolis, MN, USA). An ExoQuick-TC® ULTRA EV Kit and ExoELISA-ULTRA CD63 Kit (System Biosciences, Palo Alto, CA, USA) were used for extracellular vesicle isolation and quantification, respectively. For the semiquantitative detection of 23 human proteins in cell culture media, the human cytokine antibody array C1 (AAH-CYT-1; RayBiotech, Norcross, GA, USA), was used (Supplementary Figure S1 ). All determinations were performed according to the manufacturer’s instructions.
Aah cyt 1
Manufactured by RayBiotech
Sourced in United States
The AAH-CYT-1 is a piece of lab equipment designed for cytokine detection and analysis. It uses a proprietary technology to accurately measure the levels of various cytokines in biological samples. The core function of the AAH-CYT-1 is to facilitate cytokine quantification and profiling.
Automatically generated - may contain errors
3 protocols using aah cyt 1
1
Extracellular Vesicle Isolation and Quantification
2
Antibody Array Analysis of Conditioned Media
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Conditioned media for antibody array analysis were prepared by washing cells with PBS and incubating them in basal medium for 48 h. The conditioned media were collected in a centrifuge tube, and the cells remaining on the dish were counted to normalize conditioned media volumes by cell number. The conditioned media were clarified by brief centrifugation, 0.2 µm filtered, diluted with a serum-free medium to a concentration equivalent to 1.35 × 105 cells per 1.3 ml, and applied to the antibody arrays (Raybiotech; AAH-CYT-1) as described previously by Freund et al.19 (link) and as recommended by the supplier. The signals were detected using a G-BOX Imaging System (GeneSys) and were analyzed using specific software (GeneTools, Syngene). Signals were averaged and displayed as described in the figure legend.
3
Protein Expression and Cytokine Analysis
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Whole cell lysates were prepared using MCL1 lysis buffer in the presence of protease and phosphatase cocktail inhibitor (Sigma-Aldrich, St Louis, MO) following the manufacter's protocol. Protein lysates were subjected to SDS-PAGE on 10% polyacrylamide gel and transferred onto PVDF membranes. Membranes were probed for the protein levels of E-cadherin, and α-tubulin as loading control, using speci c primary antibodies (#4065, Cell Signaling Tech. Inc., Danvers, MA, and #sc-5286, Santa Cruz Biotechnology, Inc., Dallas, TX, respectively). Speci c bands for each protein were visualized by WesternBright™ kit (Advansta, San Jose, CA).
Enzyme-linked Immunosorbent Assay (ELISA) and human cytokine array.
Culture supernatant was collected from each growth condition, and keratinocyte growth factor (KGF-7) concentration was determined using Quantikine human KGF/FGF-7 Immunoassay (R&D Systems Inc., Minneapolis, MN). ExoQuick-TC® ULTRA EV Kit and ExoELISA-ULTRA CD63 Kit (System Biosciences, Palo Alto, CA) were used for extracellular vesicles isolation and quanti cation, respectively. For the semiquantitative detection of 23 human protein in cell culture media, the human cytokine antibody array C1 (AAH-CYT-1; RayBiotech, Norcross, GA), was used (Supplementary Figure 2). All determinations were performed according to the manufacturer's instructions.
Enzyme-linked Immunosorbent Assay (ELISA) and human cytokine array.
Culture supernatant was collected from each growth condition, and keratinocyte growth factor (KGF-7) concentration was determined using Quantikine human KGF/FGF-7 Immunoassay (R&D Systems Inc., Minneapolis, MN). ExoQuick-TC® ULTRA EV Kit and ExoELISA-ULTRA CD63 Kit (System Biosciences, Palo Alto, CA) were used for extracellular vesicles isolation and quanti cation, respectively. For the semiquantitative detection of 23 human protein in cell culture media, the human cytokine antibody array C1 (AAH-CYT-1; RayBiotech, Norcross, GA), was used (Supplementary Figure 2). All determinations were performed according to the manufacturer's instructions.
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