Live human peripheral blood mononuclear cells were labeled with Cell Proliferation Tracer Kit (Fluorometric, Blue 520). Cells cultured in medium containing Human Interleukin-2 (hIL-2) #8907 (50 ng/mL) were then left untreated (blue solid line) or stimulated with anti-CD3 (10 μg/mL) and anti-CD28 (5 μg/ml) (green solid line). Cells were analyzed by flow cytometry after 72 hr. Unstained cells were also acquired for comparison (blue dashed line).
Cell division tracking in live Jurkat cells over the course of 4 days (d0-d3). Cells were labeled with Cell Proliferation Tracer Kit (Fluorometric, Blue 520) on day 0, and analyzed by flow cytometry each day. Each successively dimmer peak represents one cell division. Unstained cells are represented by the unshaded peak.
|Product Includes||Quantity (with Count)||Solution Color|
|Cell Proliferation Tracer Dye, Blue 520||1 x 1 ea|
|Anhydrous DMSO||1 x 150 µl|
NOTE: The following protocol is a general labeling procedure. Because of differences in cell types and variations in culture conditions, optimization of the dye concentration, staining time, and/or staining temperature may be necessary. Higher dye concentrations may be required to track more cell generations, while lower concentrations may be sufficient to track fewer divisions. We recommend using the lowest dye concentration that yields sufficient signal for your assay, because cell proliferation dyes can be toxic to cells at high concentrations.
NOTE: Cell Proliferation Tracer dyes are susceptible to hydrolysis. Therefore, the DMSO stock solution should only be prepared on the day of use, and not subjected to freeze/thaw cycles. The dyes should only be added to aqueous buffer immediately before staining. Do not use buffers containing Tris or other free amines.
Additional Reagents (Not Supplied):
Note: Staining can be performed in cell culture medium containing serum, however, this results in 5-10 fold lower fluorescent signal compared to labeling in buffer without serum or other proteins.
posted January 2020
Protocol Id: 1953
The Cell Proliferation Tracer Kit (Fluorometric, Blue 520) contains Cell Proliferation Tracer Dye, Blue 520 that diffuses passively into live cells and is used for long-term cell labeling. This dye is initially a non-fluorescent ester, but is converted to a fluorescent dye by intracellular esterases. The dye then covalently reacts with amine groups on proteins, forming fluorescent conjugates that are retained in the cell. Immediately after staining, a single, bright fluorescent population will be detected by flow cytometry. Each cell division that occurs after labeling results in the appearance of a dimmer fluorescent peak on a flow cytometry histogram. The Cell Proliferation Tracer Kit (Fluorometric, Blue 520) can be used to track cell divisions in vivo or in vitro. Staining can withstand fixation and permeabilization for subsequent immunostaining.
Due to their inherent metabolic stability once inside a cell, fluorescent proliferation dyes partition in an equal manner between daughter cells during the M phase of the cell cycle. This allows the principle of dye dilution to be leveraged as a means to trace multiple rounds of cell proliferation using flow cytometry. Added benefits of proliferation dyes are that they are non-radioactive and do not require cells to be actively synthesizing DNA for efficient uptake (1-3).
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