abcam：Indirectflowcytometry(FACS)protocol.doc

Indirect flow cytometry (FACS) protocol
General procedure for flow cytometry using a primary antibody and conjugated secondary antibody
Indirect labelling requires two incubation steps, firstly with a primary antibody then with a compatible secondary antibody. The secondary (and not the primary) antibody has the fluorescent dye (FITC, PE, Cy5, etc.) conjugated. Please note that this is a general protocol and you may need to adapt it for your applications.
General Procedure:
1. Harvest and wash the cells then determine the total cell number.
Cells are usually stained in polystyrene round bottom 12 x 75 mm 2 Falcon tubes. However, they can be stained in any container for which you have an appropriate centrifuge e.g. test tubes, eppendorf tubes, and 96 well round bottomed microtiter plates. In general,cells should be spun down hard enough that the supernatant fluid can be removed with little loss of cells, but not so hard that the cells are difficult to resuspend.
It is always useful to check the viability of the cells which should be around 95% not less than 90%.
2. Resuspend the cells to approximately 1-5 x 106 cells/ml in ice cold PBS, 10% FCS, 1% sodium azide.
Use ice cold reagents/solutions and at 4oC as 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 4oC in the dark.
6. Wash the cells 3-times 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 (according to the manufacturer’s instructions) and then resuspend the cells in this solution.
8. Incubate for at least 20-30 minutes at room temperature of 4oC. This incubation must be done in the dark.
9. Wash the cells 3 X by centrifugation at 400 g for 5 min and resuspend them in ice cold PBS, 3% BSA, 1% sodium azide.
11. Store the cell suspension immediately at 4°C in the dark.
12. Analysis: For best results, analyze the cells on the flow cytometer as soon as possible.
We recommend analysis on the same day. For extended storage (16 hr) as well as for greater flexibility in planning time on the cytometer, resuspend cells in 1% paraformaldehyde to prevent deterioration.
FIXATION:
If you need to wait longer than 1 hour before analysis, you may need to fix the cells after step 5. This can preserve them for several days (this will stabilize the light scatter and inactivate most biohazardous agents). Controls will required fixation using the same procedure. Cells should not be fixed if they need to remain viable. There are several methods available. The fixation for different antigens will require optimization by the user.
1. Paraformaldehyde 0.01% to 1% for 10-15 minutes only, 100 µl per sample.
2. Acetone or methanol:
N/B polystyrene/plastic tubes are not suitable for use with acetone
Add 1ml ice cold acetone to each sample.
Mix gently. Place at -20oC for 5-10 minutes.
Centrifuge, wash twice in PBS 1% BSA.

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.

Flow cytometry intracellular staining protocol
General procedure describing detection of intracellular proteins in flow cytometry
Fixing and permeabilization:
For intracellular staining, cells can be fixed first to ensure stability of soluble antigens or antigens with a short half life (see the special recomendations below for important exceptions). This should retain the target protein in the original cellular location.
Detection of intracellular antigens requires a cell permeabilization step prior to staining. Antibodies should be prepared in permeabilization buffer to ensure the cells remain permeable. When gating on cell populations, the light scatter profiles of the cells on the flow cytometer will change considerably after permeabilization.
N/B Cell surface staining should be performed prior to fixation.
There are several methods available:
1. Formaldehyde followed by detergent:
Fixation in 0.01% formaldehyde for 10-15 min (this will stabilize proteins), followed by disruption of membrane by detergent.
Detergents:
Triton or NP-40 (0.1 to 1% in PBS). These will also partially dissolve the nuclear membrane and are therefore very suitable for nuclear antigen staining. It should be noted that loss of cell membrane and cytoplasm will result in decreased light scattering and also in reduced non specific fluorescence.
Tween 20, Saponin, Digitonin and Leucoperm are mild membrane solubilisers. Use at 0.5% v/v in PBS. These give large enough pores for antibodies to go through without dissolving plasma membrane. Suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane. Also suitable for soluble nuclear antigens.
2.  Formaldehyde (0.01%) followed by methanol  (SEE 3)
3. Methanol followed by detergent.
Add 1 ml ice cold methanol to each sample.
Mix gently. Place at -20oC for 10 minutes.
Centrifuge, wash twice in PBS 1% BSA.
4. Acetone fixation and permeabilization:
Add 1 ml ice cold acetone to each sample.
Mix gently. Place at -20oC for t5 to 10 minutes.
Centrifuge, wash twice in PBS 1% BSA.
N/B Polystyrene/plastic tubes are not suitable for use with acetone.
SPECIAL RECOMMENDATIONS:
Antigens close to the plasma membrane and soluble cytoplasmic antigens will require mild cell permeabilization without fixation.
Cytoskeletal, viral and some enzyme antigens  usually give optimal results when fixed with acetone, alcohol or formaldehyde (high concentration).
Antigens in cytomplasmic organelles and granules will require a fixation and permeabilization method depending on the antigen. The epitope needs to remain accessible.
Intracellular staining procedure:
1. Add 100 µl of fixative. Incubate for 10 minutes at required temperature (see above).
2. Add 100µl detergent based permeabilizing agent and incubate in the dark at room at room temperature for 15 minutes.
3. Wash the cells by adding 2ml of PBS (containing 0.1% triton or other permeabilizing detergent), centrifuge at 300g (2000 rpm) for 5 minutes, discard supernatant and re-suspend the pellet in the volume remaining.
4. Follow antibody staining procedure as indicated in our ‘direct’ and ‘indirect’ protocols.
Antibodies should be prepared in permeabilization buffer to ensure the cells remain permeable.
Detection of secreted proteins:
Detection of secreted proteins is difficult as the protein will be released from the cell before detection, or may degrade rapidly. Brefaldin A and other compounds are often used as a Golgi-Block. Cells are incubated with Brefaldin A which prevents proteins being released from the golgi.  Any cells expressing the protein can then be detected.