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APOPTOSIS ASSAYS
Mounting Medium. Mix 9 parts glycerol to 1 part
PBS.
NON-FACS ASSAYS
DAPI Staining
4,6-diamidino-2-phenylindole-2-HCl (DAPI) [Sigma,
D8417] binds dsDNA providing a blue fluorescence
when viewed under ultraviolet light. Apoptotic cells
are indicated by small, condensed nuclei.
Adherent cells. Plate the cells to be treated onto
sterile coverslips in a 35 mm dish. After treatment
(e.g., transfection), wash the treated cells with 1 ml
PBS (CMF) for 5 minutes. Aspirate the wash and add
1 ml Permeabilization Buffer and incubate for 10
minutes. Wash the cells with 1 ml PBS (CMF) for 5
minutes and fix in 1 ml 2% Paraformaldehyde PBS
Buffer containing 10 µg of DAPI for 10 minutes in
the dark. Wash the cells with 1 ml PBS (CMF) for 5
minutes. Mount the coverslips (cell-side down) on
slides with Mounting Medium. The coverslip can be
glued using clear nail polish. Apoptotic cells can be
observed with a fluorescent microscope at an
excitation wavelength of 350nm.
If cells are trypsinized, include the culture medium as
apoptotic cells may have detached from the bottom.
Pellet the cells (1100 rpm for 10 minutes). Aspirate
the medium and resuspend the cells in 1 ml
PBS(CMF). Pellet the cells and add 1 ml
Permeabilization Buffer. Incubate the cells for 10
minutes with gentle rocking. Pellet the cells and wash
with 1 ml PBS (CMF). Pellet the cells and fix in 1 ml
2% Paraformaldehyde PBS Buffer containing 10 µg
of DAPI for 10 minutes in the dark. Pellet the cells
and wash with 1 ml PBS (CMF) for 5 minutes. Add
an aliquot to a clean slide and mount a coverslip.
The coverslip can be glued using clear nail polish.
Apoptotic cells can be observed with a fluorescent
microscope at an excitation wavelength of 350nm.
Suspension cells. Treat suspension cells as described
for trypsinized adherent cells.
Reagents:
DAPI Stain. (Sigma, D8417). Prepare a 5 mg/ml
solution in 200 µl N,N-Dimethyl formamide (DMF).
Store in 10 µl aliquots at -20°C.
Jackson Lab
Paraformaldehyde PBS Buffer. Prepare a 2%
paraformaldehyde solution in PBS (CMF).
Permeabilization Buffer. PBS (CMF)/0.01 M
glycine/0.1% Triton X-100
DNA Laddering
Pellet treated and control cells (include culture fluid
and scraped cells if adherent). Aspirate culture fluid
and resuspend the cells in 1 ml PBS. Transfer the
cells to a 1.5 ml tube. Wash (X2) with PBS. After
final wash, pellet the cells at 14,000 rpm for 15
seconds. Aspirated the supernatant. The pellet can be
stored at -80°C until ready to assay.
DNA Isolation. Resuspend cells in Lysis buffer (1
ml/107 cells). [Typical volume is 400 µl]. Incubate
the lysate at 50°C overnight. Add 10 µl DNase-Free
RNase (50 mg/ml) and incubate for an additional 3
hours at 50°C. Extract (X1) the lysate with 1:1
phenol:chloroform. Precipitate the DNA with ½
volume 10M ammonium acetate and two volumes
absolute ethanol. Incubate at -80°C for 30 minutes.
Centrifuge the DNA for 15 minutes at 14,000 rpm.
Wash the pellet with ethanol. Decant the wash. Allow
the pellet to air dry. Resuspend the DNA in TE and
determine the concentration (A260). Store the DNA
at 4°C.
Gel electrophoresis. Heat DNA to 65°C for 10
minutes. Quench on ice. Run equal concentrations of
DNA in a 1.5% agarose gel to visualize the DNA
ladder. Runs should be long to allow good fragment
separation.
Reagents:
Lysis Buffer. Prepare fresh from stock solutions.
Volume
Stock
100 µl
250 µl
250 µl
250 µl
4150 µl
0.5 M EDTA
1 M Tris-HCl, pH 8.0
10% SDS
10 mg/ml Proteinase K
dH2O
Final
Concentration
10 mM
50 mM
0.5%
0.5 mg/ml
June 2010
FACS ASSAYS
7-AAD Staining
Wash treated cells once with PBS (Ca++, Mg++ free).
Pellet the cells and resuspend in 1 ml FACS-buffer.
Transfer the cells to a 1.5 ml tube. Pellet the cells and
resuspend in 50 ml 7-AAD staining solution.
Incubate the cells for 30 minutes in the dark at 4°C.
Pellet the cells and wash (X2) with 1 ml FACSbuffer. Pellet the cells and fix in 0.5 ml FIX-buffer.
Analyze after one hour or store at 4°C overnight in
the dark.
PI Hypotonic Buffer. Mix 50 µg/ml propidium iodide
in 0.1% sodium citrate.0.1% Triton X-100. Store in
the dark at 4°C.
Nicoletti, I, G Migilorati, MC Pagliacci, F Grignani,
and C Riccardi (1991). A rapic and simple method
for measuring thymocyte apoptosis in propidium
iodide staining and flow cytometry. J. Immunol.
Meth. 139,271.
Reagents:
7-AAD (7-amino actinomycin D, Sigma A9600).
Prepare as a 1 mg/ml (100X) stock in DMSO.
7-AAD staining solution. FACS-buffer containing 20
µg/ml 7-AAD (1/50 dilution of 100X stock).
FACS-buffer. PBS (CMF)/5% FBS/0.1% sodium
azide. Filter sterilize.
FIX-buffer. PBS (CMF)/1% paraformaldehyde/10
µg/ml 7-AAD (1/100 dilution of 100X stock).
Rabinovitch, PS, RM Torres, and D Engel (1986).
Simultaneous cell cycle and two-color surface
immunofluorescence using 7-amino-actinomycin D
and single laser excitation: Application to study cell
activation and the cell cycle of murine LY-1B cells.
J. Immunol. 136(8),2769.
Propidium Iodide Staining
Prepare cells by washing (X2) with PBS. On final
wash resuspend the cells in 1 ml PI Hypotonic buffer.
Store lysed cells in the dark at 4°C until ready to
analyze.
FACS Analysis. Analyze nuclei for PI staining and
size. Apoptotic nuclei will be smaller than controls
due to nuclear condensation. For cell cycle analysis,
determine the cellular DNA content using CELLFIT
or other appropriate statistical model.
Reagents:
Jackson Lab
June 2010