Introduction

The CellTrace™ Violet Cell Proliferation Kit provides a versatile and well-retained cell tracing reagent in a convenient and easy-to-use form. The kit contains CellTrace™ Violet in nine single-use vials to permit small scale experiments without preparing excess quantities of stock solution. CellTrace™ Violet easily diffuses into cells where it is cleaved by intracellular esterases to yield a highly fluorescent compound. This compound covalently binds to intracellular amines, resulting in stable, well-retained fluorescent staining that can be fixed with aldehyde fixatives. Excess unconjugated reagent passively diffuses to the extracellular medium, where it can be quenched with complete media and washed away.

This protocol describes a method to track the proliferation of human lymphocytes that have been stimulated with mouse anti-human CD3 and Interleukin-2, Concanavalin A, or stimulated using the Dynabeads® Human T-Activator CD3⁄CD28 for cell expansion and activation (Figure 1).  In both experiments the lymphocytes are first labeled with CellTrace™ Violet Cell Proliferation reagent.   Typically, these stimuli result in cell division every 18-20 hours.

Human peripheral blood lymphocytes were harvested and stained with CellTrace™ Violet Figure 1. Human peripheral blood lymphocytes were harvested and stained with CellTrace™ Violet. The violet peaks represent successive generations of cells stimulated with mouse anti-human CD3 and Interleukin-2 and grown in culture for 7 days. The peak outlined in black represents cells that were grown in culture for 7 days with no stimulus.
TOP

Materials

Invitrogen Materials

  • OpTmizer™ T-Cell Expansion SFM (Cat. No. 08-0022SA)
  • 200 mM L-glutamine solution (Cat. No. 25030-164)
  • Dynabeads® Human T-Expander CD3/CD28 (Cat. No. 111-41D or 111-31D)
  • GIBCO® heat-inactivated FBS (Cat. No. 10438-034)
  • Molecular Probes® CellTrace™ Violet Cell Proliferation Kit (Cat. No. C34557)
  • GIBCO® DPBS -Mg -Ca (Cat. No. 14190-144)
  • GIBCO®  1xPBS (Cat. No. 70011-044)
  • Recombinant Human Interleukin-2 (Cat. No. PHC0026)
  • Countess® Automated Counter and reagents


Additional Materials

  • 20 mL of heparinized peripheral whole blood
  • 100x Penicillin-Streptomycin Solution (Sigma)
  • GE Ficoll-Paque Plus
  • Concanavalin A lyophilized powder (Sigma)


Culture Media Preparation

To 1 L OpTmizer T-Cell Expansion SFM Basal Media, add the following:

  • 26 mL of T-Cell Expansion Supplement (supplied in Cat. No. 08-0022SA)
  • 10 mL of 200 mM L-glutamine solution (final conc. 2 mM).
  • 10 mL of Penn/Strep solution (100x) (100,000 units penicillin, 100 mg streptomycin)
  • Complete media is stable for 4 weeks when stored at  2-8°C in the dark.


Interleukin-2 (IL-2) Stock Solution

  • Dilute 5 µL glacial (17M) acetic acid into 800 µL dH2O to make a 100 mM stock solution.
  • Dissolve 40 µg IL-2 in 400 µL acetic acid to make 0.1 mg/ml solution.
  • Aliquot 20 µL into microfuge tubes and store at -20ºC.
  • 1 µL of this solution contains 100 ng IL-2.


Concanavalin A Stock Solution

  • Dissolve 20 mg of Concanavalin A into 10 mL of PBS to make a 2mg/mL stock solution.
  • This solution is stable at 4°C for several months.


CD3 Concentration

  • 0.5 mL bottle of Invitrogen CD3, Mouse Anti-Human, (Purified) ( MHCD0300) contains 100 µg anti-CD3 antibody.
  • 1 µL of stock contains 200 ng antibody.
TOP

Dynabeads® Wash Procedure

  1. Resuspend the Dynabead® samples in the vial.

  2. Transfer the desired volume of Dynabead® samples to a tube.

  3. Add the same volume of PBS, or at least 1 mL, and mix.

  4. Place the tube in a DynaMag™-15 magnet for 1 minute and discard the supernatant.

  5. Remove the tube from the magnet and resuspend the washed Dynabead® samples in the same volume of PBS as the initial volume of Dynabead®  samples.

NOTE: Please see Dynabeads® Human T-Activator CD3⁄CD28 for Cell Expansion and Activation for a more detailed protocol.

TOP

Mononuclear Cell Isolation

1.  Dilute 20 mL whole blood in 20mL 1XPBS and mix well.

2. Add 15 mL Ficoll-Paque Plus to a 50 mL centrifuge tube, layer 20 mL diluted whole blood on top.

3. Centrifuge 30 minutes at 400 x g; carefully remove lymphocyte layer.

4.  Resuspend cells in 25 mL DPBS buffer in a 50 mL conical tube.

5.  Spin 5 minutes at 300 x g, pour off supernatant, and resuspend in 25  mL DPBS.

6.  Repeat wash step and resuspend in 10 mL DPBS.

7.  Count cells on the Countess® Automated Counter or by another method;  adjust concentration to 1x106 cells/mL.


TOP

Cell Staining

Reserve 1 mL of cells as unstained control.

1.  Stain 10 mL of cells with 20 µl 5mM CellTrace™ Violet (10µM final concentration).

2.  Immediately vortex  for 30 seconds.

3. Place tubes on rocker, covered, for 20 minutes.

4.  Add 2 mL cold FBS and incubate 5 minutes.

5.  Wash cells once with DPBS + 10% FBS.

6.  Resuspend cells in 10 mL OpTmizer T-Cell Expansion media.

7  Distribute 1 mL aliquots of stained cells into a culture plate or flask.

8. Stimulate each 1 mL aliquot of cells with one of the following treatments:

      a.    200 ng anti-CD3 (2uL), incubate 30 minutes; add 100 ng IL-2 (1 µl) in culture media.

      b.    5 µg/mL ConA (2.5 µl of 2 mg/mL solution)

      c.    50 µl CD3/CD28 T cell expander beads (2 beads per cell)


9.  Incubate for desired length of time at  37º/5% CO2. (These stimuli typically result in cell division every 18-20 hours).
TOP

Analysis

After staining with CellTrace™ Violet dye, reserve a 500 µL aliquot for initial analysis. Re-evaluate cells again after 7 days, or when sufficient growth has occurred. For each analysis, cell should be stained with a dead cell stain such as Sytox® Red dye or Sytox® AADvanced™ dye and appropriate monoclonal antibodies to distinguish live T Cells. Additional antibodies or functional dyes can be used with or without fixation to further characterize cells.

TOP

References

  1.    J Cell Biol 101, 610 (1985).

  2.    J Cell Biol 103, 2649 (1986).

  3.    J Immunol Methods 171, 131 (1994).

  4.    J Exp Med 184, 277 (1996).

  5.    J Immunol Methods 133, 87 (1990).

  6.    Transplant Proc 24, 2820 (1992).

  7.    Current Protocols in Cytometry, J. P. Robinson, Ed., (1998) pp 9.11.1-9.11.9.

TOP
MP 34557      11–Jan–2010