FACS - General Tips/Protocols and Troubleshooting

FACS (Flow cytometry) is a widely used method for analyzing expression of cell surface and intracellular molecules, characterizing and defining different cell types in heterogeneous cell populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells. It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins or ligands that bind to specific cell-associated molecules, such as DNA binding by propidium iodide. The staining procedure involves making a single-cell suspension from cell culture or tissue samples. The cells are incubated in tubes or microtiter plates with unlabeled or fluorochrome-labeled antibodies and then analyzed on the flow cytometer.

FACS - General Tips/Protocols

Direct Cell Surface Staining Protocol

Direct staining is done with a primary labeled antibody.

General Procedure:

1.	Harvest, wash the cells and adjust cell suspension to a concentration of 1-5x10⁶ cells/ml in ice cold PBS, 10% FCS, 1% sodium azide.
	Note: Use ice cold reagents/solutions and at 4°C. Low temperature and presence of sodium azide prevent the modulation and internalization of surface antigens, which can produce a loss of fluorescence intensity.
2.	Add 0.1-10 µg/ml of the primary labeled antibody. Dilutions, if necessary, should be made in 3% BSA/PBS (Propridium iodide can also be added at this point for dead cell exclusion).
3.	Incubate for at least 30 min at room temperature or 4°C. This step will require optimisation.
4.	Wash the cells 3x by centrifugation at 400 g for 5 minutes and resuspend them in 500 µl to 1 ml of ice cold PBS, 10% FCS,1% sodium azide.
5.	Keep the cells in the dark on ice or at 4°C in a fridge until your scheduled time for analysis.
6.	Analysis: For best results, analyze the cells on the flow cytometer as soon as possible.

Indirect Cell Surface Staining Protocol

Indirect staining requires two incubation steps: the first with a primary antibody followed by a compatible labeled secondary antibody.

General Procedure:

1.	Harvest, wash the cells and determine the total cell number.
2.	Resuspend the cells to approximately 1-5x10⁶ cells/ml in ice cold PBS, 10% FCS, 1% sodium azide.
	Note: Use ice cold reagents/solutions and at 4°C. Low temperature and presence of sodium azide prevent the modulation and internalization of surface antigens, which can produce a loss of fluorescence intensity.
3.	Add 100 µl of cell suspension to each tube.
4.	Add 0.1-10 µg/ml of the primary antibody. Dilutions, if necessary, should be made in 3% BSA/PBS.
5.	Incubate for at least 30 min at room temperature or 4°C in the dark.
6.	Wash the cells 3x by centrifugation at 400 g for 5 min and resuspend them in ice cold PBS. You may need to adjust the conditions of the centrifugation (the force and the time) for the cell types used.
7.	Dilute the fluorochrome-labeled secondary antibody in 3% BSA/PBS at the optimal dilution and then resuspend the cells in this solution.
8.	Incubate for at least 20/30 min at room temperature of 4°C. This incubation must be done in the dark.
9.	Wash the cells 3x by centrifugation at 400 g for 5 min and resuspend them in ice cold PBS, 3% BSA, 1% sodium azide.
10.	Store the cell suspension immediately at 4°C in the dark.
11.	Analysis: For best results, analyze the cells on the flow cytometer as soon as possible.

Note: Do not add sodium azide to buffers if you are concerned with recovering cell function, e.g. if cells are to be collected for functional assays. It inhibits metabolic activity.

FACS - Troubleshooting Tips

No signal/weak fluorescence intensity

Signal not correctly compensated
Check if positive single color control is set up correctly on flow cytometer and gated/compensated correctly to capture all the events.

Insufficient antibody present for detection
Increase amount/concentration of antibody.

Target protein not present/expressed at low level
Ensure tissue/cell type expresses target protein and that it is present in a high enough amount to detect.

Fluorochrome fluorescence has faded
Antibody may have been kept for too long or left out in the light. Fresh antibody will be required.

The primary antibody and the secondary antibody are not compatible
Use secondary antibody that was raised against the species in which the primary was raised.

High fluorescence intensity

Antibody concentration too high
This will give high non-specific binding or very high intensity of fluorescence. Reduce the amount of antibody added to each sample.

Inadequate blocking
Add 1% to 3% blocking agent with antibody as well as a blocking step.

High background/high percentage of positive cells

Gain set too high/offset too low
Use the positive control to set up the flow cytometer correctly again, using the offset to reduce background from small particles and reduce the gain to decrease the signal.

Excess antibody
Decrease the antibody concentration. You can also add detergent to the wash buffers to ensure washing away of excess antibody.

Two or more cell populations observed when there should be just one

Cell doublets present
Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot. Mix cells gently before staining and again before running on the cytometer using a pipette.

High side scatter background (from small particles)

Cells lysed
Ensure cells in the sample have not lysed and broken up. Samples should be fresh and prepared correctly.

Bacterial contamination
Ensure sample is not contaminated. Bacteria will auto fluoresce at low level. This will also give a high event rate.

Low event rate

Low number of cells/ml
Run 1x10⁶ cells/ml. Ensure cells are mixed well (but gently).

High event rate

High number of cells
Dilute to between 1x10⁵ and 1x10⁶ cells/ml.

FACS - References

Flow Cytometry Protocols (3^rd Edition): T.S. Hawley & R.G. Hawley; Methods Mol. Biol. 699, 1-485 (2011)

FACS - Online Resources

http://en.wikipedia.org/wiki/Flow_cytometry

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