Spleen B cells were isolated (B cell isolation kit, Miltenyi) and cultured overnight in the presence of 25 μg/ml LPS (Escherichia coli 055:B5, Sigma-Aldrich, Diegem, Belgium) to maintain sufficient cell survival and support antigen presentation. Dead cells were removed by Ficoll centrifugation (Lympholyte-M, Cedarlane Labs, Atlanta, GA, USA) and remaining B cells were stained with Cyto-ID Red long-term cell tracer kit (Enzo Life Sciences, Lausen, Switzerland) following manufacturers’ instructions. B cells were then cocultured for 18 h with CD4+ T cells (ratio B:T, 1:5) in the presence of indicated peptide (2 μM, added to the culture media). Annexin V APC was used to detect cell death in B cells (Annexin V detection kit, BD Biosciences) according to manufacturers’ instructions. Gated B cells were then analyzed for Annexin V binding on flow cytometer. For inhibition of granzyme-B (GZB) activity, Z-AAD-CMK (Calbiochem/Merck, Overijse, Belgium) was added at 20 μg/ml during the entire coculture period. Inhibition of FasL was performed with functional grade anti-mouse CD178 antibody (clone MFL3, eBioscience) at 20 μg/ml during the coculture period.
Cyto id red long term cell tracer kit
Manufactured by Enzo Life Sciences
Sourced in Switzerland, Taiwan, Province of China, United States
The Cyto-ID Red long-term cell tracer kit is a fluorescent dye-based solution used for tracking and monitoring cell populations over extended periods. The dye binds to cellular components, allowing for the visualization and identification of labeled cells through fluorescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
B cell isolation kit, by Miltenyi Biotec (1 mentions)
Annexin 5 detection kit, by BD (1 mentions)
Lps escherichia coli 055 b5, by Merck Group (1 mentions)
Lympholyte m, by Cedarlane (1 mentions)
Zaad cmk, by Merck Group (1 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
Incucyte caspase 3 7 dye, by Sartorius (1 mentions)
Incucyte live cell analysis system, by Sartorius (1 mentions)
4 protocols using cyto id red long term cell tracer kit
1
Evaluating B Cell Apoptosis in Presence of CD4+ T Cells
2
Tracking Cell Migration in PLGA Scaffolds
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The Cyto-ID™ Red Long-Term Cell Tracer Kit (ENZO Life Sciences, Taipei, Taiwan) was used to label a red fluorescent dye containing hydrophobic aliphatic chains into the cell membrane’s lipid bilayer for tracking cell migration, because PLGA is a nontransparent material. In brief, cells were collected from the culture flask and the suspended cells were stained with cell tracker dye for 5 min, according to the user guide. After centrifuging to remove excess dye, the cells were resuspended in culture medium. Before seeding the cells, nine grids with an area of 1 cm2 were marked in the bottom of a 10 cm2 cell culture dish. Then, the samples got stuck in the bottom of the dish and aligned with the grids. A concentration of 8,000 cells/well of the suspended cell solution was added to the flat, micro/nano, and microfibrous scaffold, respectively. After incubation for 24 h, an inverted fluorescence microscope (Leica) was used to record cell migration image for 24 h. The ImageJ software was applied to measure the distance of cell migration.
3
Cytotoxicity Assay of Tumor Cells
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Killing of A431 epidermoid carcinoma cells by CD8 T cells or NK cells was measured using an Incucyte Live-Cell Analysis System (Sartorius). Target cells were labeled with the CYTO-ID Red Long-Term Cell Tracer Kit (Enzo Life Sciences, #ENZ-51037-K025) and co-incubated with prestimulated or resting PBMC (for NK cell killing assays) or prestimulated enriched CD8 T cells to a defined effector-to-target ratio (optimal 10:1). Incucyte Caspase-3/7 dye (Sartorius, #4440) was added to the co-cultures to determine target cell apoptosis induced by NK cells or CD8 T cells. Live cell images for time-course experiments were taken every 2 hours. Target cell death was quantified via the overlap area between the Caspase-3/7 dye and the target cell counterstain (total object area in µm/image).
4
In vivo Tracking of hPMSCs
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To track hPMSC in vivo, hPMSCs were first labelled using CytoID red long-term cell tracer kit (Enzo Life Science; USA). Briefly, cells were trypsinized and labelled with 1 ml of 2x CytoID for 5 min. Staining was stopped by adding 2 ml of stop buffer. Cells were centrifuged (400 g, 5 min), cell pellets resuspended in 10 ml of complete media (DMEM+10% FBS+1% P/S) in a T75 flask and incubated at 37 °C for at least 12 h. CytoID-labelled hPMSCs were prepared and injected as described.
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