test the effect of Roscovitine | My Assignment Tutor

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: MicroscopePipetteTrypan BlueTrypsinFCSEmpty tubesHemocytometerPipette 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:  DeadViable (Alive)Top Left1935Top Right55120Middle41129Bottom Left3371Bottom Right1035 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:  DeadViable (Alive)Top Left75151Top Right1749Middle2118Bottom Left1932Bottom Right2135 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:  DeadViable (Alive)Top Left1732Top Right1521Middle3225Bottom Left2511Bottom Right2842 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:  DeadViable (Alive)Top Left2220Top Right3111Middle255Bottom Left2810Bottom Right258 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:  DeadViable (Alive)Top Left2518Top Right158Middle2510Bottom Left2210Bottom Right353 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:  DeadViable (Alive)Top Left325Top Right253Middle223Bottom Left285Bottom Right301 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.

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