CellTrace™ CFSE Cell Proliferation Kit

实验概要

The CellTrace™  CFSE Cell Proliferation Kit provides a versatile and well-retained  cell-tracing reagent in a convenient and easy-to-use form. The kit  contains carboxyfluorescein diacetate succinimidyl ester (CFDA-SE),  often called CFSE, in ten single use vials. Small-scale experiments can  be performed without preparing excess quantities of perishable CFSE  stock solution. For additional convenience, we include high-quality DMSO  (dimethylsulfoxide) and a detailed protocol.

CFSE passively diffuses into cells. It is colorless and  nonfluorescent until the acetate groups are cleaved by intracellular  esterases to yield highly fluorescent carboxyfluorescein succinimidyl  ester. The succinimidyl ester group reacts with intracellular amines,  forming fluorescent conjugates that are well retained and can be fixed  with aldehyde fixatives. Excess unconjugated reagent and by-products  passively diffuse to the extracellular medium, where they can be washed  away.

The dye–protein adducts that form in labeled cells are retained by  the cells throughout development and meiosis, and can be used for in  vivo tracing. The label is inherited by daughter cells after either cell  division or cell fusion, and is not transferred to adjacent cells in a  population.Lymphocytes labeled with CFSE have been detected up to eight  weeks after injection into mice in lymphocyte-migration studies, and  viable hepatocytes that were similarly labeled were easily located by  fluorescence microscopy even 20 days after intrahepatic transplantation.

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.

1. Resuspend cells of interest in prewarmed PBS/0.1% BSA at a final concentration of 1 × 10⁶ cells/mL.

2. For most applications add 2 μL of 5 mM stock CFSE solution per mL of cells for a final working concentration of 10 μM.

3. Incubate dye at 37°C for 10 min.

4. Quench the staining by the addition of 5 volumes of ice-cold culture media to the cells.

5. Incubate 5 min on ice.

6. Pellet cells by centrifugation.

7. 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.

8. Set up in vitro cell cultures under appropriate conditions or adoptively transfer cells.⁷

9. Harvest cells and stain for other markers if appropriate.

10. Analyze using a flow cytometer with 488 nm excitation and emission filters appropriate for fluorescein.

2. Alternate Method to Label Cells in Suspension

1. Centrifuge to obtain a cell pellet and aspirate the supernatant.

2. 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).

3. Resuspend the cells gently in prewarmed (37°C) PBS containing the probe (prepared in previous step).

4. Incubate the cells for 15 min at 37°C.

5. Re-pellet the cells by centrifugation and resuspend in fresh prewarmed medium.

6. 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

1. Grow cells to desired density on coverslips inside a petri dish filled with the appropriate culture medium.

2. 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).

3. Remove the medium from the dish and add prewarmed (37°C) PBS containing the probe (prepared in previous step).

4. Incubate the cells for 15 min at 37°C.

5. 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

1. Before fixation, the cells must be washed with PBS or other suitable buffer.

2. 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.

3. After fixation, the cells should be rinsed in PBS.

4. 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.

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