Annexin V Protocol

Annexin V conjugates allow the identification of cell surface changes that occur early during the apoptotic process using flow cytometry. Early in the apoptotic process, phosphatidylserine becomes exposed on the cell surface by flipping from the inner to outer leaflet of the cytoplasmic membrane. This event is thought to be important for macrophage recognition of cells undergoing apoptosis. The binding of Annexin V to phosphatidylserine is calcium dependent, reversible, and very tight with a Kd of approximately 5 x 10^-10 M.

Notes:

Different cell types vary in their phosphatidylserine (PS) content, along with the amount of PS exposure on the cell surface after cell death. The following protocol is a guideline for getting started, however it may be necessary to adjust the concentration of the Annexin V-FITC. Typically a 1 to 100 dilution of the Annexin V-conjugate is appropriate, however dilutions of 1:10 up to 1:1000 may be needed.

When setting up an experiment, it is necessary to calibrate the flow cytometer to avoid spectral overlap between the two PMT channels. For each experiment, a set of treated cells should be processed unstained and stained separately with Annexin V-FITC and propidium iodide to define the boundaries of each population.

Protocol for Flow Cytometry:

1. Collect cells (approximately 5 x 10⁵ to 1 x 10⁶ cells per tube) by gently centrifugation at room temperature in 6-ml polystyrene Falcon tubes (#2058). Remember when using adherent cells that both the supernatant and attached cells should be collected and examined together.
2. Wash the cells once in 500ml of cold 1X PBS buffer gently resuspending the cells then pelleting by centrifugation as in step 1.
3. Per each sample of cells, prepare 100 µl of Annexin V incubation reagent by combining:
   10 ul 10X Binding Buffer
   10 ul Propidium Iodide
   1 ul Annexin V-FITC
   79 ul dH₂O
   100 µl Total
    
   Keep this cocktail in the dark and on ice.
4. Prepare 400 µl of 1X Binding Buffer per each sample for diluting the cells after incubation. 1:10 dilution in dH₂O of 10X Binding Buffer.
5. Gently resuspend the washed cells in 100 µl of the Annexin V incubation reagent prepared in step 3.
6. Incubate in the dark for 15 minutes at room temperature.
7. Add 400 µl of 1X Binding Buffer to each 100 µl reaction sample.
8. Examine by flow cytometry within 1 hour for maximal signal.

TUNEL Protocol

Notes:

This technique uses the enzyme terminal deoxynucleotidyl transferase (TdT) to label cells that have oligonucleosomal nicks/strand breaks in their DNA. PI or 7-AAD can be included for cell cycle determination, although it is very difficult to obtain tight CVs. The protocol below is carried out in 96-well V-bottomed micro titer plates (MTP); with some modifications, it could be carried out in 12 x 75 mm plastic tubes. Phoenix Flow Systems has a protocol for pre-fixing samples with PFA and ethanol, storing them, and labeling them at some later date.

Materials:

• In Situ Cell Death Detection Kit (Boehringer Mannheim 1 684 79550)
• Set aside 100µl label solution for 2 "no enzyme" controls
• Add all (50µl) of one bottle of enzyme solution to the remaining label solution (450µl) and mix to make enough reaction mixture for 10 samples
• 96 well V-bottomed microtiter plate (MTP).
• 12x75mm snap cap tubes (Falcon 2054).
• PBS + 1% BSA (Sigma A-7030).
• Fresh 4% paraformaldehyde in PBS pH 7.4
• Shaker for incubating cells in MTP.
• Permeabilization buffer: 0.1% Triton X-100 in 0.1% sodium citrate.
• Propidium Iodide (PI) buffer (optional)
• 5µg/ml PI (Sigma P-4170) and 200µg/ml DNase-free RNase A (Sigma R-5503) in PBS.

Controls Required:

• Cells only (no treatment/no stain)
• Cells + label WITHOUT enzyme
• Untreated/non-apoptotic cells with label and enzyme
• If PI has been included: one tube with untreated fixed cells in PI buffer, WITHOUT TUNEL label or enzyme

Procedure:

1. Wash cells twice in PBS/1% BSA at 4⁰
2. Resuspend to 2x10⁷ cells/ml
3. Add 100µl cells to one MTP well for each sample and control.
4. Add 100 ml fresh 4% paraformaldehyde in PBS pH 7.4 to each well
5. Resuspend thoroughly and incubate 30 minutes at room temperature WHILE SHAKING
6. Centrifuge MTP at 300xG for 10 minutes and remove fixative by flicking or suction
7. Wash once with 200µl PBS/BSA as above
8. Resuspend cells in 100µl permeabilization buffer per well for 2 minutes on ice (4⁰.)
9. Wash twice as above
10. Resuspend each sample in 50µl reaction mixture and the "no enzyme" control in 50µl label solution
11. Cover MTP and incubate 60 minutes at 37⁰ in humidified air in the dark
12. Wash twice as above
13. Resuspend each well in a 12x75mm tube with 500µl PBS/BSA OR with 500µl PI buffer
14. Analyze

PARP PE staining

1. Begin with 5 X 10⁵ cells per ml to be fixed and examined. Place the cells in a 6-ml polystyrene Falcon tube (#2058).
2. Fix the cells as follows:
   
   • Spin cells down and wash in 500 l of 1X PBS.
   • Resuspend in 500 ul Cytofix while vortexing.
   • Incubate in Cytofix for 30 minutes at RT.
   • Spin down the cells and wash in 1 mL of Perm Wash.
   • Resuspend in 1 mL of Perm Wash.
   • Incubate for 10 minutes at RT.
   • Spin down the cells and resuspend in 50ml of Perm Wash.
3. Add 10 ul PARP PE
4. Incubate in the dark for 30 minutes at room temperature.
5. Add 450 µl of Perm Wash solution.
6. Examine by flow cytometry.