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Protocol
In this section there are 3 experiments in which you can test your technique or get ideas about your own experiments. These experiments show reliable and consistent results and are relatively easy to perform.
Sample Experiment #1: Human Cell Line
Materials
- U937 cell line
- ATCC CRL-1593.2
- Human interferon-gamma (IFN-γ) and interleukin-4 (IL-4)
- BD # 554617 and BD # 554605
- Formaldehyde: 16% solution in water, ampules
- Electron Microscopy Sciences (# 15710)
- 100% 4°C methanol
- Staining media:
- PBS
- 0.5% BSA
- 0.02% NaN3
- Phospho-specific antibodies
- Becton Dickinson
- Phospho-Stat1 (Y701) clone 4a
- Alexa Fluor 488 (BD# 612596)
- Phospho-Stat6 (Y641) clone 18
- Alexa Fluor 647 (BD # 612601)
- 5 ml FACS tubes
- Culture/Stimulation Media:
- RPMI
- 10% FBS
- PSG
Methods
Stimulation:
Sample |
1 |
2 |
3 |
4 |
5 |
Stimulus |
None |
None |
IFN-γ |
IL-4 |
IFN-γ + IL-4 |
Concen. |
--- |
--- |
10 ng/ml |
10 ng/ml |
10 ng/ml; 10ng/ml |
1. Suspend cells at ~1x106 cells/mL in culture media, and warm to 37°C in a tissue culture incubator (5% CO2). Aliquot 1 ml (~1x106 cells) per stimulation condition into FACS tubes.
2. Add IFN-γ and/or IL-4 at a final concentration of 10 ng/mL (e.g. 10 μL of a 1 μg/mL cytokine stock).
3. Vortex to mix. Incubate for 15 min at 37 °C, 5% CO2.
Fixation and Permeabilization:
1. Add formaldehyde directly to the cells at a final concentration of 1.5% (1:10 dilution of the 16% stock; ~100µl for 1ml sample), and vortex to mix. Do this quickly for all samples. The media will turn yellow.
2. Incubate for 10 min at room temperature.
3. Pellet the cells by centrifugation at 500g for 5 min at 4°C. Do not add any staining media prior to the spin; it is bad for the methanol permeabilization step.
4. Decant supernatant, and vortex to thoroughly resuspend cells in the residual buffer. Then add 4°C MeOH (about 1 ml per 1x106 cells) and vortex.. The addition of methanol to a pellet of cells will result in severe clumping and cell loss.
5. Permeabilize at 4°C for 10 min, -20°C overnight, or -80°C longer than overnight. Signal decreases slightly with long permeabilization periods. The permeabilization is the best place to take an overnight break (rather than after the staining).
Staining and Analysis:
Sample |
1 |
2 |
3 |
4 |
5 |
Stain |
none |
pStat1-Alexa488 + pStat6-Alexa647 |
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1. Add 3 ml staining media to samples. Pellet the cells as in Step 3.
2. Wash the pellet with ~3 ml staining media. Pellet.
3. Resuspend cells at ~1x107/ml (100 µl for 1x106 cells) in staining media.
4. Pipet 100 µl of each sample into a new tube. Identical staining volumes are critical for consistent staining between samples.
5. Add the indicated antibodies (20 ul each) to the samples. In order to achieve consistent staining, make up a cocktail of the antibodies needed for all the samples in one vial, then distribute the antibodies to the samples.
6. Incubate for 30 min at room temperature.
7. Wash with 3 ml staining media.
8. Analyze on FACSCalibur style instrument with 488 and 633 nm laser lines.
a. Set PMTs so that unstained cells appear in the lower quadrant of both channels.
Expected Results:
- Unstained/Unstimulated: lowest staining in both channels.
- Unstimulated: low staining in both channels, slightly higher than the unstained sample.
- IFN-γ stimulation: high pStat1 staining. IFN-γ is a strong inducer of pStat1. pStat6 staining should remain as in sample 2. Fold change in pStat1 (calculated as [median fluorescence intensity (MFI) of stimulated cells]/[MFI of unstimulated cells]) should be 10-fold or greater.
- IL-4 stimulation: high pStat6 staining, low pStat1 staining (as in sample 2). IL-4 induces pStat6 selectively. Fold change in pStat6 should be ~5-fold.
- Dual stimulation: high pStat1 and pStat6 staining. Fold changes should be identical to those observed in samples 3 and 4.
