实验材料

Kit Contents

• CellTrace™ CFSE (Component A), 10 vials, each containing 50 μg of lyophilized powder
• DMSO (Component B), 1 vial containing 0.5 mL of high quality dimethylsulfoxide

Materials Needed but Not Included:

• Cells of interest as a single-cell suspension
• PBS/0.1% BSA
• 5 mM stock solution of CFSE probe (see Reagent Preparation)
• Culture media for cells of interest
• Flow cytometer with 488 nm argon-ion laser

Storage and Handling

Upon receipt, components should be stored desiccated at ≤–20°C until required for use. AVOID REPEATED FREEZING AND THAWING. Before opening the vial, allow the product to warm to room temperature. When stored properly, both the DMSO and solid CFSE should be stable for at least six months. Solutions of the reagent should be used promptly.

Spectral Characteristics

The approximate excitation and emission peaks of this product after hydrolysis are 492 nm and 517 nm, respectively. Cells labeled with CellTrace™ CFSE can be visualized by fluorescence microscopy using standard fluorescein filter sets or analyzed by flow cytometry in an instrument equipped with a 488 nm excitation source.

实验步骤

For staining cells prior to flow cytometric analysis of cell proliferation or cell division, the protocol below is appropriate. More information on this procedure can be found in reference 7. Our suggested initial conditions may require modifications because of differences in cell types and culture conditions.

The concentration of probe for optimal staining will vary depending upon the application; we recommend testing at least a tenfold range of concentrations. In general, long-term staining (more than about three days) or the use of rapidly dividing cells will require 5–10 μM dye. Less dye (0.5–5 μM) is needed for shorter experiments, such as viability assays. Microscopy applications may require up to 25 μM CFSE. To maintain normal cellular physiology and reduce potential artifacts from overloading, the concentration of dye should be kept as low as feasible.

Note: The CellTrace™ CFSE dye reacts with amine groups and should not be used with amine-containing buffers or lysine-coated slides.

Reagent Preparation

Prepare a 5 mM CellTrace™ CFSE stock solution immediately prior to use by dissolving the contents of one vial (Component A) in 18 μL of the DMSO provided (Component B).

1. Labeling Cells for Analysis in Flow Cytometry

This method has been useful in determining cell division in B and T cells. Note: To ensure uniform labeling, it is important that you begin with a single-cell suspension (no aggregates). The quantity of cells for in vitro labeling experiments is usually 105 –106 , depending upon how long after labeling then cells will be allowed to grow. For adoptive transfers, label from 1–5 × 107 cells.

Resuspend cells of interest in prewarmed PBS/0.1% BSA at a final concentration of 1 × 106 cells/mL. For most applications add 2 μL of 5 mM stock CFSE solution per mL of cells for a final working concentration of 10 μM. Incubate dye at 37°C for 10 min. Quench the staining by the addition of 5 volumes of ice-cold culture media to the cells. Incubate 5 min on ice. Pellet cells by centrifugation. Wash the cells by resuspending the pellet in fresh media. Pellet and resuspend the cells in fresh media a further two times for a total of three washes. Set up in vitro cell cultures under appropriate conditions or adoptively transfer cells.7 Harvest cells and stain for other markers if appropriate. Analyze using a flow cytometer with 488 nm excitation and emission filters appropriate for fluorescein.

2. Alternate Method to Label Cells in Suspension

Centrifuge to obtain a cell pellet and aspirate the supernatant. Dilute the 5 mM CFSE stock solution in phosphate-buffered saline (PBS) or other suitable buffer to the desired working concentration (0.5–25 μM). Resuspend the cells gently in prewarmed (37°C) PBS containing the probe (prepared in previous step). Incubate the cells for 15 min at 37°C. Re-pellet the cells by centrifugation and resuspend in fresh prewarmed medium. Incubate the cells for another 30 min to ensure complete modification of the probe and then wash the cells again.

3. Alternate Method to Label Adherent Cells

Grow cells to desired density on coverslips inside a petri dish filled with the appropriate culture medium. Dilute the 5 mM CFSE stock solution in phosphate-buffered saline (PBS) or other suitable buffer to the desired working concentration (0.5–25 μM). Remove the medium from the dish and add prewarmed (37°C) PBS containing the probe (prepared in previous step). Incubate the cells for 15 min at 37°C. Replace the loading solution with fresh, prewarmed medium and incubate the cultures for another 30 min at 37°C. During this time, CFSE will undergo acetate hydrolysis.

4. Optional Fixation and Permeabilization

Before fixation, the cells must be washed with PBS or other suitable buffer. Standard fixation protocols using aldehyde-containing fixatives should effectively crosslink the amines of the protein–probe conjugate. Typically, cells are fixed for 15 min at room temperature using 3.7% formaldehyde. After fixation, the cells should be rinsed in PBS. If needed, cells can be permeabilized by any appropriate protocol (for example, 10 minute incubation in ice-cold acetone). Following permeabilization, the cells should be rinsed in PBS. Permeabilization is required, for example, if the cells are to be subsequently labeled with an antibody.