FACS Procedures for Apoptosis Detection
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Materials:
-
Hoechst 33258 (Sigma B-2883).
- stock: 10 mg/ml in dH20 (40 )
- working dilution: 500µg/ml (50µl stock + 950µl PBS).
-
7-Amino-actinomycin (Sigma A-9400)
- stock: 200µg/ml in dH20 (40 )
- 12x75mm snap cap tubes (Falcon 2054).
- Cells only (no treatment/no stain)
- Treated cells with Hoechst only
- Treated cells with 7-AAD only
- Untreated cells with both stains
- Resuspend at least 0.5 x 106 cells in 12x75mm tubes at a concentration of 106 cells/ml.
-
Add Ho 33258 at a final concentration of 1µg/ml to samples.
- 2µl/ml of working solution per tube.
- Incubate the samples in a 370 waterbath for 7 minutes.
- Remove samples from the waterbath and immediately place on ice.
-
Add 7-AAD at a final concentration of 1µg/ml to samples.
- 5µl/ml of stock solution per tube.
- Incubate on ice for 10 minutes.
- Analyze samples.
- Primary laser: 310nm (UV), 50mW.
- Secondary laser: 488nm (visible), 250mW.
- Ho 33258 detection: 424/44 BP filter, Fl-1 detector.
- 7-AAD detection: 660/20 BP filter, Fl-4 detector.
<center> <h3> <a name="brduapop"> TUNEL</a></h3><a name="brduapop"> </a><a name="brduapop"> </a></center> In Situ Cell Death Detection Kit from Boehringer Mannheim , which uses directly labeled dUTP-FITC. Other protocols call for BrdU and FITC-anti BrdU , biotinylated dUTP and streptavidin-FITC, etc. 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.
- 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
- Wash cells twice in PBS/1% BSA at 40 .
- Resuspend to 2x107 cells/ml.
- Add 100µl cells to one MTP well for each sample and control.
- Add 100 ul fresh 4% paraformaldehyde in PBS pH 7.4 to each well.
- Resuspend thoroughly and incubate 30 minutes at room temperature WHILE SHAKING.
- Centrifuge MTP at 300xG for 10 minutes and remove fixative by flicking or suction.
- Wash once with 200µl PBS/BSA as above.
- Resuspend cells in 100µl permeabilization buffer per well for 2 minutes on ice (40 .)
- Wash twice as above.
- Resuspend each sample in 50µl reaction mixture and the "no enzyme" control in 50µl label solution.
- Cover MTP and incubate 60 minutes at 370 in humidified air in the dark.
- Wash twice as above.
- Resuspend each well in a 12x75mm tube with 500µl PBS/BSA OR with 500µl PI buffer
- Analyze.
<center><a name="brduapop"> </a><h3><a name="brduapop"> </a><a name="annexin"> <b>Annexin V + 7-AAD</b> </a></h3><a name="annexin"> </a><a name="annexin"> </a></center> 7-AAD for the PI provided with the kit.
Materials:
-
Apoptosis Detection Kit (R&D Systems KNX50) .
- contains: FITC-conjugated human annexin V, PI, 10x Binding Buffer.
-
7-Amino-actinomycin (Sigma A-9400)
- stock 200µg/ml in dH20 (40 ) .
- 12x75mm snap cap tubes (Falcon 2054).
- PBS.
- Cells only (no treatment/no stain)
- Treated cells with FITC-Annexin V only
- Treated cells with 7-AAD only
- Untreated cells with both stains
- Resuspend at least 0.5 x 106 cells in 12x75mm tubes at a concentration of 106 cells/ml.
- Add 2ml of cold PBS to tubes.
- Centrifuge for 8 minutes at 1800rpm.
- Resuspend pellet in 2ml of cold PBS.
- Centrifuge for 8 minutes at 1800rpm.
- Resuspend cells in 0.1ml of 1x binding buffer.
- Add 10µl of FITC-conjugated annexin V and 5µl of 7-AAD to tubes.
- Gently vortex.
- Incubate at room temperature for 15 minutes in the dark.
- Add 0.9ml of 1x binding buffer.
- Analyze samples within 1 hour of staining.
- Primary laser: 488nm (visible), 250mW.
- FITC-annexin V detection: 530/20 BP filter, Fl-1 detector.
- 7-AAD detection: 660/20 BP filter, Fl-3 detector.