ATDC5 cells were treated with 5 µg/ml LPS for 5 h after 48 h of transfection. Then, cells were digested using 0.25% trypsin, followed by washing with PBS. Cells were subsequently fixed with 70% ethanol overnight at 4°C. Apoptosis was detected using the Annexin V-FITC- propidium iodide kit (cat. no. 70-AP101-100; Multisciences Lianke Biotech Co., Ltd.) according to the manufacturer's protocol. Cell apoptosis rate was measured using a FACSCalibur flow cytometer (BD Biosciences) with Cell Quest software version 5.1 (BD Biosciences). The experiment was performed in triplicate.
Cell quest software version
Manufactured by BD
The Cell Quest software is a flow cytometry data acquisition and analysis software. It provides core functionality for setting up and controlling flow cytometry instruments, as well as for analyzing the collected data.
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Lab products found in correlation
2 protocols using cell quest software version
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Apoptosis Quantification in LPS-treated ATDC5 Cells
2
Pathway Analysis and Cell Cycle Profiling
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The association between phenotypes, pathways and USP18/Skp2 expression was analyzed using GSEA v2.2 (http://www.broad. mit.edu/gsea/). GSEA calculates a gene set Enrichment Score that estimates whether genes from a pre-defined gene set (obtained from the Molecular Signatures Database, MSigDB, http://software.broadinstitute.org/gsea/msigdb/ index.jsp) are enriched among the highest-or lowest-ranked genes or distributed randomly. Default settings were used. Thresholds for significance were determined by permutation analysis (1,000 permutations). false discovery rate (FDR) was calculated. A gene set was considered significantly enriched when the FDR score was <0.05.
Cell cycle analysis. Cells transfected with USP18 or empty vectors were fixed in 70% ethanol for 12 h at 4˚C, treated with 0.1 mg/ml RNase A for 30 min at 37˚C, and stained with 50 mg/ml propidium iodide for 30 min at 37˚C in the dark (PI; BD Biosciences, Franklin Lakes, NJ, USA). The cells were then analyzed by flow cytometry using FACSCalibur (BD Biosciences) (13) , and cell cycle distributions were calculated using CellQuest software version 5.1 (BD Biosciences). These experiments were performed three times in triplicate.
Cell cycle analysis. Cells transfected with USP18 or empty vectors were fixed in 70% ethanol for 12 h at 4˚C, treated with 0.1 mg/ml RNase A for 30 min at 37˚C, and stained with 50 mg/ml propidium iodide for 30 min at 37˚C in the dark (PI; BD Biosciences, Franklin Lakes, NJ, USA). The cells were then analyzed by flow cytometry using FACSCalibur (BD Biosciences) (13) , and cell cycle distributions were calculated using CellQuest software version 5.1 (BD Biosciences). These experiments were performed three times in triplicate.
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