實驗概要
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.
實驗材料
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.
實驗步驟
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.
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.
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).
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