Materials Provided

CFSE dye (2 vials, 500 μg dye per vial)

Materials needed but not provided

· Viable single-cell suspensions of primary lymphoid cells of interest

· Fresh cell culture-grade Dimethyl Sulfoxide (DMSO; eg, Sigma D2650)

· Sterile Dulbecco"s Phosphate Buffered Saline (1× DPBS)

· Cell culture medium, eg, with 10% Fetal Bovine Serum (FBS)

· Sterile polypropylene 15-mL or 50-mL conical tubes

· Microcentrifuge tubes or 2-mL cryovials

· Fluorescent antibodies for immunophenotypic or functional analysis (optional)

Required Equipment

· Blue laser-equipped cytometer; eg, BD FACSCanto™ II, BD LSRFortessa™, BD LSR™ II Flow Cytometer, or BD Accuri™ C6

· Centrifuge

· 37°C CO2 Incubator and 37ºC Water Bath

Storage

Upon arrival, store the dye powder with desiccant and protected from light at ≤ -20°C until use. After reconstitution with DMSO, store the stock solution with desiccant at ≤ -20°C in small aliquots. The stock solution is stable for 3 months if stored as indicated.

Procedure

CFSE has a maximum absorption of 490 nm and a maximum emission of 520 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the emitted fluorescence.

Preparation of CFSE dye solution in DMSO

1.Prepare a 10 mM stock solution of CFSE dye by adding 90 μL DMSO to one vial of CFSE. Mix thoroughly by vortexing.

2.Prepare single-use aliquots of the CFSE stock solution (user-defined volume) using appropriately labeled microcentrifuge tubes or 2-mL cryovials.

3.Freeze all aliquots at ≤ -20°C with desiccant and protected from light.

CFSE labeling of cells

This procedure has been optimized for labeling mouse and human lymphocytes at cell concentrations of 10-30 × 10^6 cells/mL. Assay conditions may need to be optimized for other cell types or cell densities.

1.Thaw the 10 mM stock solution of CFSE, if previously frozen.

2.Transfer cells to 15- or 50-mL polypropylene centrifuge tubes.

3.Wash cells in 1× DPBS to remove any residual serum proteins.

4.Repeat step 3.

5.Resuspend the cells thoroughly into a single cell suspension at a concentration of 10-30 × 10^6 cells/mL in 1× DPBS.

a.Note: Avoid staining in azide-, serum-, or protein-containing buffers, as these moieties bind free dye and may interfere with staining of the cells.

6.Add stock CFSE to the single cell suspension to achieve a final concentration of 1-10 μM. If necessary, the stock solution may be diluted further with DMSO before addition to the single cell suspension. Vortex immediately.

7.Incubate the dye-cell suspension in a 37°C water bath for 10-15 minutes.

8.Add 9× the original volume of 1× DPBS to the cells and pellet cells by centrifugation.

9.Decant the supernatant and gently mix to disrupt the cell pellet.

10.Add 10 mL of complete medium with 10% FBS and repeat centrifugation step.

11.Decant supernatant and gently mix to disrupt the cell pellet.

12.Resuspend the cells in complete medium and proceed to cell culture or analysis by flow cytometry.

Notes:

1.Confirm brightness of the cell labeling by comparing labeled cells with unlabeled control cells.

2.BD Horizon™ CFSE can be used in intracellular staining assays that require fixation with formaldehyde and permeabilization with methanol and detergents such as those used for BD Phosflow™ staining (eg, Cat. No. 558050, BD Phosflow™ Perm Buffer III), intracellular cytokine staining (eg, Cat. No. 554714, BD Cytofix/Cytoperm™ Fixation/Permeablization Kit), or transcription factor staining (eg, Cat. No. 562574/562725, BD Pharmingen™ Transcription Factor Buffer Set).

3.Higher initial staining intensities can be achieved by increasing the initial CFSE dye loading concentration. However, as with other succinimidyl ester containing tracking dyes, higher loading concentrations may lead to increased cell toxicity or cell death.