CAFs were seeded in 96-well plates at a density of 1.0 3 10 3 cells/well and incubated at 37 C with 5% CO 2 . At 24 hours after seeding, the CAFs were exposed to pro-inflammatory cytokines (IL1a, IL1b, and TNFa). OCUM-2MD3 cells were seeded into 6-well plates at a density of 2.0 3 10 5 cells/well and incubated at 37 C with 5% CO 2 . At 24 hours after seeding, cells were exposed to either S-CAF-CM or CAF-CM for 48 hours. Cell proliferation was then measured with a Luna cell counting plate or using an IncuCyte S3 Livecell Analysis system (Essen BioScience). For the analysis with the IncuCyte system, CAFs were seeded into 96-well plates and stained with Nuc-Light Rapid Red (Essen BioScience). The plate was inserted into the IncuCyte instrument for real-time imaging, with four fields imaged per well every 24 hours over 10 days. Data were analyzed using the IncuCyte software, which quantified the percentage of red confluence values. All groups were tested in triplicate, and the data are presented as the means ± standard errors of the means (SEM).
Nuc light rapid red
Manufactured by Sartorius
The Nuc-Light Rapid Red is a laboratory equipment designed for the rapid detection and quantification of nucleic acids. It utilizes a fluorescence-based method to provide a fast and accurate assessment of nucleic acid content in samples.
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Lab products found in correlation
2 protocols using nuc light rapid red
1
Effects of Inflammatory Cytokines on CAF-Mediated Cancer Cell Proliferation
2
Cytotoxic CD8+ T cell-mediated OPC death
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OPCs were stained with an eFluor 633 dye prior to the addition of CD8s to ensure subsequent quantification was restricted to the OPC population. Unstained OT-1 CD8s were isolated and added to the stimulated and stained OPCs. Cell membrane permeable, caspase 3/7 Green reagent (1:1000; Essen Biosciences) was added to determine caspase activity. The caspase 3/7 reported was designed to report on caspase 3/7 activity by targeted cleavage of the DEVD peptide sequence and subsequent nuclear translocation of the linked DNA binding dye (Essen Biosciences). Images were captured every 2–3 h using IncuCyte S3 Live Cell Analysis System (Sartorius). Cultures were analyzed for caspase 3/7 stain colocalization with NucLight Rapid Red (Essen Biosciences) labeled OPCs and the applied mask was counted as particles per image with 9 images taken. A total of three experiments were completed. MHC class I inhibitors were applied two hours before antigen was given to IFNγ stimulated OPCs and kept in culture for the antigen loading portion of the experiment until CD8s were applied and antigen, IFNγ and inhibitors were washed away from the culture. Fas/FasL and Granzyme B inhibitors were applied to the culture at the time of OPC-CD8 co-culture initiation and were kept in culture for the remainder of the experiment.
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