Fluorescent Labelling Lab Notebook (ELN 5)
Instructions
- Lab notebooks are completed digitally using the Crowdmark platform. You will submit your lab notebook before the end of the lab time.
- The amount of work expected for these lab notebooks is reflective of the time available! Labs run for 3 hours – we have therefore designed the lab activities and the lab notebook prompts in such a way that they will be able to be completed within the 3 hour window of provided time.
- It is strongly advised that you read the lab outline BEFORE the lab starts to ensure you will be prepared to manage your time effectively and complete all protocols during the lab.
- This lab notebook will be completed as PAIRS
- This lab notebook (and completion of the lab) is pass/fail
- Assignment Deadlines
- Policies on Submitting Work, Absences and Missed Work
- Specifications guidelines for Electronic Lab Notebooks
Part 1: Pre-lab
- What is the purpose of today’s lab activity?
- What protocols will you be completing in the lab?
- Are there any safety precautions to be aware of before starting?
Part 2: Cell Concentration and Passaging Calculations
Last week you had completed cell counts using a haemocytometer. Go back to the cell count data you collected in Part 1 of Cell Biology (it was submitted as part of ELN 4). Use your raw data from Table I to complete the calculations below and answer the questions. The calculation questions should be answered in order, as some steps rely on the answers to the previous step. Show your work (and all units) for each calculation. The video below might help with understanding some of the calculation steps (starting at 4:10):
Option A: Use the textbox on Crowdmark to show your calculations.
- Math nomenclature using normal keyboard symbols when typing:
- * can be used to represent multiplication
- / can be used to represent division
- () and [] are extra useful for keeping numerators and denominators of fractions (separated with a / for division) clear.
- x can be used to indicate that the value is in standard notation, e.g. 2.8x10^5 cell/ml
- ^ can be indicated directly before an exponent value, e.g. 2.8×10^5 cells/ml
- expression of denominators could be as a fraction (e.g. cells/ml) or with a negative exponent (e.g. cells * ml^-1)
Option B: Open a MS word document and use the equation editor there. Then, you will take a screenshot of the calculations, and input the screenshot, rather than writing out your steps in the text box on Crowdmark.
Option C: Complete your analysis freehand on a blank sheet of paper. You can hand this in to your TA with your partner names clearly indicated at the top if you would prefer showing your work on paper instead of digitally.
Option D: Complete your analysis within the same excel spreadsheet where your Table I is stored. Make it clear to your TA that you have shown your work inside the same excel spreadsheet, and then re-upload the revised Table I excel file saved as a .pdf file.
- Calculate and report the cell viability (% of viable cells) in your simulated cell suspension.
- Cell Viability: Cell viability (% of viable cells) = (# of viable cells)/(# of viable cells + # of dead cells) x 100%
- Calculate and report the cell concentration (# of viable cells per ml) as determined by your haemocytometer counts.
- Cell Concentration: To calculate the cell concentration in a haemocytometer (viable cells per ml), you need to consider that each square is a rectangular prism with a length, width and height as follows: 1 mm x 1 mm x 0.1 mm. Therefore, you know how many cells per rectangular prism, which has a known volume (in mm3). Note: 1 mm3 = 1 μL. Use this as a guide to assess if you calculated the volume correctly. Once you determine how many ml are in the rectangular prism, you can determine the # of cells in 1.5 ml (this was the total volume of cell suspension that you removed from the petri dish during the cell passaging activity last week).This value can then be multiplied by the total dilution factor to determine the # of viable cells per ml in the cell suspension (Remember: When using a haemocytometer, occasionally cell suspensions have to be diluted because the cell concentration is too high to do a proper count. There is also the consideration of what happens to the cell concentration when trypan blue is added).
- Report the recommended seeding density (viable cells/cm2) for the C2C12 cell line according to the American Type Culture Collection (ATCC).
- Calculate and report the surface area (in cm2) of a 60 mm diameter cell culture plate.
- Calculate and report how many mls of your cell suspension should be added to a 60 mm cell culture plate to achieve the seeding density recommended by ATCC.
- How many mls of cell culture media should be added to this dish (assuming the final volume in the dish is 5 ml)?
- Recall that the total volume in the dish will be composed of some of your cell suspension and the remainder of the space will be filled with fresh cell culture media. How can you determine how much media to top the plate up with if you know the total volume and how much cell suspension you have already added?
- Based on the answers above, what would be the split ratio for this plate? Assume the total volume of cell suspension was 1.5 ml.
- (Note: round your split ratio to the nearest whole number please! 1:X , where X is the rounded whole number.)
- HINT: If you added 0.3 ml of the total volume of cell suspension (1.5 ml) into a new plate, this would be a 1:5 split ratio (i.e. you added 1/5 of the cell suspension into a new plate).
Part 3: Experimental Results
in a few sentences, provide some observations about the cells you observed under the fluorescent inverted microscope. Consider how the cells looked in normal light as well as how they looked using the fluorescent light to view just the mitochondria. Did you notice a qualitative difference between the control cells and the cells treated with hydrogen peroxide? Describe the difference.
