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53452
Cell Proliferation Tracer Kit, (Fluorometric, Blue 520)
Cellular Assay Kits
Assay Kit

Cell Proliferation Tracer Kit, (Fluorometric, Blue 520) #53452

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Flow Cytometry Image 1: Cell Proliferation Tracer Kit, (Fluorometric, Blue 520)

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).

Flow Cytometry Image 2: Cell Proliferation Tracer Kit, (Fluorometric, Blue 520)

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.

To Purchase # 53452S
製品番号 サイズ 価格 在庫
53452S
1 Kit

Product Includes Quantity (with Count) Solution Color
Cell Proliferation Tracer Dye, Blue 520 1 x 1 ea
Anhydrous DMSO 1 x 150 µl

Protocol

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Cell Proliferation Tracer Kit, (Fluorometric, Blue 520) #53452

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.

A. Solutions and Reagents

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.

Supplied Reagents:

  • Cell Proliferation Tracer Dye, Blue 520 (#73595S): Prepare a cell proliferation dye stock solution by dissolving one vial of Cell Proliferation Tracer Dye in 20µL of anhydrous DMSO. This brings the stock solution concentration to 5mM. Protect dye stock solutions from light.
  • Anhydrous DMSO (#15360S)

Additional Reagents (Not Supplied):

  • 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100mL 10X PBS (#12528) to 900mL water, mix.

B. Labeling of Target Cells

  1. Pellet cells by centrifugation and aspirate the supernatant.
  2. Resuspend cells at 106 cells/mL in pre-warmed (37°C) PBS (or similar buffer) containing 1uM cell proliferation dye. Protect cells from light for this and all subsequent steps.

    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.

  3. Incubate the cells for 10-15 minutes at room temperature or 37°C, to allow dye uptake.
  4. Add an equal volume of cell culture medium and incubate for 5 minutes at room temperature or 37°C to hydrolyze free dye.
  5. Pellet the cells by centrifugation and resuspend in an equal volume of fresh pre-warmed cell culture medium.
  6. Incubate the cells for 15-30 minutes at 37°C to allow the dye to react with intracellular proteins.
  7. Pellet the cells by centrifugation and resuspend in an equal volume of fresh pre-warmed cell culture medium. Proceed to flow cytometry analysis (step 9). Alternatively, return cells to incubator and culture for the desired period of time to allow cells to divide.
  8. Optional: perform formaldehyde fixation, permeabilization, and/or immunostaining.
  9. Analyze by flow cytometry in the appropriate channel.

posted January 2020

Protocol Id: 1953

Product Description

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.

Background

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).

  1. Wallace, P.K. et al. (2008) Cytometry A 73, 1019-34.
  2. Quah, B.J. and Parish, C.R. (2012) J Immunol Methods 379, 1-14.
  3. Tario, J.D. et al. (2011) Methods Mol Biol 699, 119-64.

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