Detection of Apoptosis by a Tunel Technique

1. Harvest 1.5 x 106 cells and wash in PBS.

2. Resuspend in 1 mL of PBS, add 1 mL 2% w/v paraformaldehyde, and place on ice for 15 min.

3. Wash twice in PBS and resuspend the pellet in 2 mL of 70% ethanol.

4. Place at -20°C for at least 30 min, but overnight (approx 16 h) may give better results. Cells can be stored for up to 3 d at this stage.

5. Rehydrate the cells in PBS by pelleting the cells, aspirating the ethanol, and resus-pending the cells in 1 mL of PBS.

6. Pellet and resuspend the cells in 50 |xL of cacodylate buffer (0.2 M potassium cacodylate, 2.5 mM Tris-HCL, pH 6.6, 2.5 mM CoCl2, 0.25 mg/mL bovine serum albumin, 5 units of TdT, and 0.5 nMdUTP-FITC) and incubate for 60 min at 37°C.

7. Wash twice in PBS.

8. Analyze on a histogram plot of green fluorescence against cell numbers.

Note: A control sample should omit Tdt in Step 6.

Propidium Iodide 0 Propidium Jodide

0 Propidium Iodide 1^3 0 Propidium Iodide 1023

Fig. 2. Propidium iodide/RNAse-stained cell lines showing (upper panels) unperturbed cell cycle distributions and (lower panels) sub G0/Gx apoptotic populations in the Mx interval gate. HL-60 cells are shown on the left panels, and K562 cells are shown on the right.

0 Propidium Iodide 1^3 0 Propidium Iodide 1023

Fig. 2. Propidium iodide/RNAse-stained cell lines showing (upper panels) unperturbed cell cycle distributions and (lower panels) sub G0/Gx apoptotic populations in the Mx interval gate. HL-60 cells are shown on the left panels, and K562 cells are shown on the right.

5.3. Detection of PS as a Marker of Apoptosis Using Annexin V (Fig. 3)

1. Wash 1 X 106 cells in PBS and resuspend in 1 mL of incubation buffer (10 mM HEPES, pH 7.4, 150 mMNaCl, and 5 mMCaCl2).

2. Add FITC-labeled annexin V to a final concentration of 2.5 |j,g/mL.

3. Wash once in incubation buffer, resuspend in 1 mL of incubation buffer, and analyze cells using a histogram of green fluorescence on the x-axis and cell numbers on the y-axis.

4. Use nonstained cells and untreated cells as negative controls.

5. Alternatively, if PI (5 |j,g/mL) is incorporated in the incubation buffer at step 4, a dot plot of PI on the x-axis against annexin V on the y-axis can be used to distinguish viable cells (which are negative for both PI and annexin V), apoptotic cells (which are annexin V-positive but exclude PI and are therefore PI-negative), and late apoptotic or necrotic cells (which are double-positive for PI uptake and annexin V staining).

Fig. 3. (A) FITC-conjugated annexin V staining for phosphatidylserine exposure on apoptotic cells (right) compared with viable control cells (left). (Images were kindly provided by Ulrike Jahnke, Barts and The London School of Medicine and Dentistry.) (B) Dual-staining of control cells (left) and apoptotic cells (right) stained with prop-idium iodide (PI) (x-axis) and annexin V (y-axis). The quadrant analysis shows viable cells negative for annexin V and excluding PI (lower left). Apoptotic cells stain with annexin V but excluding PI (upper left). Secondary necrotic cells (i.e., necrosis after apoptosis) are positive for both PI and annexin V (upper right). Necrotic or mechanically damaged cells positive for PI only are shown in the lower right quadrant (very few).

Fig. 3. (A) FITC-conjugated annexin V staining for phosphatidylserine exposure on apoptotic cells (right) compared with viable control cells (left). (Images were kindly provided by Ulrike Jahnke, Barts and The London School of Medicine and Dentistry.) (B) Dual-staining of control cells (left) and apoptotic cells (right) stained with prop-idium iodide (PI) (x-axis) and annexin V (y-axis). The quadrant analysis shows viable cells negative for annexin V and excluding PI (lower left). Apoptotic cells stain with annexin V but excluding PI (upper left). Secondary necrotic cells (i.e., necrosis after apoptosis) are positive for both PI and annexin V (upper right). Necrotic or mechanically damaged cells positive for PI only are shown in the lower right quadrant (very few).

5.4. Detection of Changes in Aym Using Nonfixable Dyes (Fig. 4A)

1. Harvest 1 X 105 cells and wash once in PBS.

2. Resuspend cells in 1 mL medium containing 40 nM DiOC6 or alternatively 1 ^.M JC-1.

3. Incubate cells for 15 min at 37°C.

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