Practical 6
Aim: To test the effect of Roscovitine for a long period of time (7 days) with L6 cells and short period of time (1hr)
Materials:
- Microscope
- Pipette
- Trypan Blue
- Trypsin
- FCS
- Empty tubes
- Hemocytometer
- Pipette Aid (Gun)
- Glass Graduated Pipette
Methods:
Staining Cells and Saving Photos:
1. Check wells to see which wells has the more live cells.
2. Well 2 was treated with CT for a long period of time (7days), wells 3 treated with CT for 1hour (short period) and well 1 was used as my control.
3. Then add 1uL of NucBlue to wells 1, 2 and 3. Give it a bit of mix before placing under the microscope.
4. Place wells under the microscope and save photo of wells 1, 2 and 3.
Cell Counting using Hemocytometer:
1. Set up microscope.
2. Pipette 10 uL of Trypan Blue and 10 uL of diluted cells from wells 1, 2 and 3 into separate tubes.
3. Record and calculate the numbers for dead and live cells. Empty wells 1, 2 and 3 for cell counting.
4. Add 100 uL of Trypsin and leave it for 5 mins.
5. Add 50 uL of FSA.
6. Prepare hemocytometer for cell counting.
Results:
Calculations:
C1= 1mg/ ml 1mg/ml X 50uL / 1000 uL
V1= 50uL = 0.05 mg/ mL
C2= 0.05mg/ml = 50ug/mL
V2= 1000 uL
Treated L6 cells with Roscovitin at a concentration of 50ug/mL.
Control Well 1
Upper Area:
| Dead | Viable (Alive) | |
| Top Left | 19 | 35 |
| Top Right | 55 | 120 |
| Middle | 41 | 129 |
| Bottom Left | 33 | 71 |
| Bottom Right | 10 | 35 |
1. % of cells: 2. Average of cells/ square
390 X 100= 71.167% 390 = 78
548 5
3. Dilution Factor 4. Concentration (viable cell/mL)
20 =2uL 548 x 2 x 104= 10,960,000
10
Lower Area:
| Dead | Viable (Alive) | |
| Top Left | 75 | 151 |
| Top Right | 17 | 49 |
| Middle | 21 | 18 |
| Bottom Left | 19 | 32 |
| Bottom Right | 21 | 35 |
1. % of cells: 2. Average of cells/ square
285 X 100 = 65.068% 285 = 57
438 5
3. Dilution Factor 4. Concentration (viable cell/mL)
20 = 2uL 438 X 2 X 104= 8,760,000
10
Treatment Well 2 ( 7day treated with CT)
Upper Area:
| Dead | Viable (Alive) | |
| Top Left | 17 | 32 |
| Top Right | 15 | 21 |
| Middle | 32 | 25 |
| Bottom Left | 25 | 11 |
| Bottom Right | 28 | 42 |
1. % of cells: 2. Average of cells/ square
131 x 100 = 52.822% 131 = 26.2
248 5
3. Dilution Factor 4. Concentration (viable cell/mL)
20 = 2uL 248 x 2 x 104= 4,960,000
10
Lower Area:
| Dead | Viable (Alive) | |
| Top Left | 22 | 20 |
| Top Right | 31 | 11 |
| Middle | 25 | 5 |
| Bottom Left | 28 | 10 |
| Bottom Right | 25 | 8 |
1. % of cells: 2. Average of cells/ square
54 x 100 = 29.189% 54 = 11
185 5
3. Dilution Factor 4. Concentration (viable cell/mL)
20 = 2uL 185 x 2 x 104 = 3,700,000
10
Wells 3 Treated with CT for 1 HR
Upper Area:
| Dead | Viable (Alive) | |
| Top Left | 25 | 18 |
| Top Right | 15 | 8 |
| Middle | 25 | 10 |
| Bottom Left | 22 | 10 |
| Bottom Right | 35 | 3 |
1. % of cells: 2. Average of cells/ square
49 x 100 = 29% 49 = 9.8
171 5
3. Dilution Factor 4. Concentration (viable cell/mL)
20 = 2uL 171x 2 x 104= 3,420,000
10
Lower Area:
| Dead | Viable (Alive) | |
| Top Left | 32 | 5 |
| Top Right | 25 | 3 |
| Middle | 22 | 3 |
| Bottom Left | 28 | 5 |
| Bottom Right | 30 | 1 |
1. % of cells: 2. Average of cells/ square
17 x 100 = 11% 17 = 3.4
154 5
3. Dilution Factor 4. Concentration (viable cell/mL)
20 = 2uL 154 x 2 x 104= 3,080,000
10
Control Well 1
A B
Figure 3:
(A) Confocal Microscopy image of L6 cells under the microspe without Roscovitine. Cells were stained with 1uL of Nucblue. Scale bar 54 μm.
(B) Confocal Microscopy Flourosent image of L6 cells under the microspe without Roscovitine. Cells were stained with 1uL of Nucblue. Scale bar 54 μm.
Treatment Wells 2 (7 day period)
A B
Figure 4:
(A) Confocal Microscopy image of L6 cells under the microspe treated with Roscovitine for a long period of time. Cells were stained with 1uL of Nucblue. Scale bar 54 μm.
(B) Confocal Microscopy Flourosent image of L6 cells under the microspe treated with Roscovitine for a long period of time. Cells were stained with 1uL of Nucblue. Scale bar 102 μm.
Treatment Wells 3 (1hr)
A B
Figure 5:
(A) Confocal Microscopy image of L6 cells under the microspe treated with Roscovitine for a short period of time. Cells were stained with 1uL of Nucblue. Scale bar 126 μm.
(B) Confocal Microscopy Flourosent image of L6 cells under the microspe treated with Roscovitine for a short period of time. Cells were stained with 1uL of Nucblue. Scale bar 114 μm.
