TITLE: Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay
AUTHORS: Catherine  M. Worsley, Rob  B. Veale, Elizabeth  S. Mayne

[DESCRIPTION]
Cell death is a key component of mammalian physiology, and can happen as a result of structural damage, or actively as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V apoptosis assay. 

[STEPS]
SECTION: Cell culture of mammalian solid tumour cells lines
1. Bring all reagents to 37°C in a waterbath.
            Note: ensure that all surfaces and equipment are cleaned with 70% ethanol. Perform all work in a laminar airflow cabinet.

SECTION: Cell culture of mammalian solid tumour cells lines
2. In a 10cm culture dish, add 10ml of Dulbecco’s Modified Eagle’s Medium (DMEM)/Hams F12 (3:1) supplemented with 10% fetal calf serum (FCS) and 2% penicillin/streptomycin.
SECTION: Cell culture of mammalian solid tumour cells lines
3. Add the cell suspension to the dish, then incubate at 37°C with humidity, and 5%C CO2 in air to mimic in vivo conditions.
SECTION: Cell detachment
4. Bring all reagents to 37°C in a waterbath.
SECTION: Cell detachment
5. Observe cell growth daily using an inverted light microscope
SECTION: Cell detachment
6. When cells reach 80% confluency, remove the culture media and wash the cells with 2ml phosphate buffered saline (PBS). Discard the PBS.
SECTION: Cell detachment
7. Add 2ml of trypsin/EDTA (TE) solution and incubate at 37°C for 5-10 mins or until cells start to detach from the culture dish. Note: do not leave cells in TE for too long as it can cause damage to the cells. To speed up detachment, lightly move the dish so that the TE covers the whole area.
SECTION: Cell counting using Trypan Blue
8. Once cells are detached, add 2ml of the cell-TE mixture to 8ml of DMEM/HAMS F12 medium.
SECTION: Cell counting using Trypan Blue
9. On a piece of parafilm, pipette 20µl of 0.4% trypan blue.
SECTION: Cell counting using Trypan Blue
10. Add 20µl of the cell mixture to the trypan blue on the parafilm, and gently mix together by slowly pipetting up and down
SECTION: Cell counting using Trypan Blue
11. Add 20µl of the cell/trypan blue mixture to the chamber of the haemocytometer, and cover with a coverslip. This will ensure that the liquid moves onto the counting chambers by capillary action. Ensure that there are no bubbles under the coverslip.
SECTION: Cell counting using Trypan Blue
12. Focus the microscope on the gridlines using the 10x objective. 
SECTION: Cell counting using Trypan Blue
13. Count the lives cells (live cells exclude trypan blue) in one set of 16 squares. 
SECTION: Cell counting using Trypan Blue
14. Move the haemocytometer to count the cells in the next set of 16 squares. Do this 4 times.
SECTION: Cell counting using Trypan Blue
15. To calculate the number of viable cells/ml:
a.Add the total number of cells together
b.Divide by the number of sets of squares counted to get the average number of cells. 
c.Multiply by the dilution factor (2)
d.Multiply by 10 000 (104) (this is the volume of the squares)
e.Calculate the volume needed to get the required number of cells per dish (in this case 150 000)
Example:
Number of cells (84)
Number of block sets (4)= 21 (average number of cells per block)

21 x dilution factor (2) x 10 000 = 420 000 cells/ml

Number of cells wanted (150 000)
Cells/ml (420 000)= 0.357ml of cell suspension to each dish
SECTION: Cell counting using Trypan Blue
16. Pipette the volume of cell suspension calculated above into the number of 6cm dishes required (7 dishes).
SECTION: Cell counting using Trypan Blue
17. Add culture medium to the dish to make the total volume up to 1ml
SECTION: Cell counting using Trypan Blue
18. Place dishes into the incubator and leave overnight at 37°C with humidity, with 5% CO2 in air.
SECTION: Cell death induction
19. Prepare the Staurosporine (STS) and pH-adjusted media.
SECTION: Cell death induction
20. STS preparation:
a.Dissolve STS powder in tissue culture grade DMSO to obtain a 1mM concentration
SECTION: Cell death induction
21. pH-adjusted media preparation:
Adjust medium pH to required pH using hydrochloric acid (HCl) and measure with a pH meter 
SECTION: Cell death induction
22. Remove all the dishes from the incubator, and wash the cells with 2ml of PBS.
SECTION: Annexin V FITC and Propidium Iodide staining
24. Remove medium (with floating cells) and place in a 15ml tube. Centrifuge at 1000rpm for 5 min.
SECTION: Annexin V FITC and Propidium Iodide staining
25. Remove the conditioned media supernatant and store at -20°C.
SECTION: Annexin V FITC and Propidium Iodide staining
26. Trypsinize adherent cells in 1 ml TE and add to a tube containing 4ml of DMEM/HAMS F12 culture media.
SECTION: Annexin V FITC and Propidium Iodide staining
27. Centrifuge at 1000rpm for 5 min. Discard the supernatant.
SECTION: Annexin V FITC and Propidium Iodide staining
28. Resuspend pellets of both tubes in PBS and combine into 1 tube.
SECTION: Annexin V FITC and Propidium Iodide staining
29. Add 5ml of PBS. Centrifuge at 1000rpm for 5 min. Discard supernatant.
SECTION: Annexin V FITC and Propidium Iodide staining
30. Resuspend cells in 1-2ml of 1x binding buffer
SECTION: Annexin V FITC and Propidium Iodide staining
31. To 100µl of cell suspension, add 5µl of Annexin V FITC and 5 µl of propidium iodide (PI)
SECTION: Annexin V FITC and Propidium Iodide staining
32. Incubate at room temperature in the dark for 15 min
SECTION: Annexin V FITC and Propidium Iodide staining
33. Add 200 µl of binding buffer and acquire on the BD LSR II flow cytometer.
SECTION: Flow Cytometer setup
34. Switch the BD LSR II flow cytometer on and open FACSDiva software. Allow the instrument to warm up for at least 30 minutes. Ensure that maintenance has been performed and that the instrument is in good working order.
SECTION: Flow Cytometer setup
35. Verify instrument performance by running BD Cytometer Setup and Tracking (CST) beads
SECTION: Flow Cytometer setup
36. Create an experiment in FACSDiva software
SECTION: Flow Cytometer setup
37. To adjust photon multiplier tube (PMT) voltages for compensation, prepare unstained and single stained tubes as follows:
a.Tube 1 - 100µl of unstained cells
b.Tube 2 - 100µl of cells stained with 5µl Annexin V FITC
c.Tube 3 - 100µl of cells stained with 5µl PI
SECTION: Flow Cytometer setup
38. Acquire unstained cells. Adjust PMT voltages to ensure that cells are in the negative quadrant for all fluorophores
Note: this needs to be done as some cells autofluoresce which can interfere with the assay
SECTION: Flow Cytometer setup
39. Acquire tube 2. Adjust PMT voltages so that there is a well separated positive and negative population in the FITC channel. Adjust PMT voltages so that all other channels only have a negative population.
SECTION: Flow Cytometer setup
40. Acquire tube 3. Adjust PMT voltages so that there is a well separated positive and negative population in the PerCP-Cy5.5 channel. Adjust PMT voltages so that all other channels only have a negative population
SECTION: Flow Cytometer setup
41. Once compensation has been set up, acquire experimental tubes
Note: acquire and record as many cells as possible for analysis. 
SECTION: Flow Cytometer setup
42. Perform maintenance and shutdown of the flow cytometer as per manufacturer’s instructions
SECTION: Analysis
43. Export the FCS data files in FSC 2.0 and 3.0 to a folder or external harddrive
SECTION: Analysis
44. Import FSC files into FlowJo software
SECTION: Analysis
45. Select the untreated control tube and double click. A forward scatter (FSC) vs. side scatter (SSC) plot should open
SECTION: Analysis
46. Select the cell population of interest and using the gating tool to draw a gate around the population. Name or label that population of cells.

 

Figure 1. FSC vs. SSC plot
SECTION: Analysis
47. Double click on that gate and another FSC/SSC plot should appear.
SECTION: Analysis
48. Click on the X–axis and change it to Annexin V or the FITC channel
SECTION: Analysis
49. Click on the Y-axis and change it to the PI or PerCP-Cy5.5 channel
SECTION: Analysis
50. Insert a quadrant or spider quadrant gate as shown below in Figure 2. Gates should separate live cells (Q4: Annexin V-/PI-), early PCD (Q3: Annexin V+/PI-), late PCD (Q2: Annexin V+/PI+) and dead/necrotic (Q1: Annexin V-/PI+) from each other. The numbers in the quadrants indicate the percentage of cells in that quadrant



 

Figure 2. Annexin V FITC vs. Propidium Iodide showing cells undergoing PCD
SECTION: Cell death induction
23. Treat the 7 dishes as follows:
 
     Dishes      Dish 1 - untreated control      Dish 2 - DMSO      Dish 3 - STS+DMSO      Dish 4 - pH 6.5 15min      Dish 5 – pH 6.5 1hr      Dish 6 – pH 3.2 15min      Dish 7 – pH 3.2 1hr        Initial steps      Add 6ml of   serum-free DMEM/Hams F12 medium      Add 10µl   DMSO per ml of culture media (i.e. 60µl DMSO in 6ml culture media)      Add 10µl   STS+DMSO per ml of culture media       Add 6ml of pH   6.5 media. Place back into the incubator for 15 minutes.      Add 6ml of pH   6.5 media. Place back into the incubator for 1 hour.      Add 6ml of pH   3.2 media. Place back into the incubator for 15 minutes.      Add 6ml of pH   3.2 media. Place back into the incubator for 1 hour.        Additional   steps      None      None      None      Remove media   after 15min and wash cells in 2ml PBS. Remove PBS and add 6ml serum-free   media      Remove media   after 1 hr and wash cells in 2ml PBS. Remove PBS and add 6ml serum-free media      Remove media   after 15min and wash cells in 2ml PBS. Remove PBS and add 6ml serum-free   media      Remove media   after 1 hr and wash cells in 2ml PBS. Remove PBS and add 6ml serum-free media        Incubation      Incubate   overnight at 37°C