DrNoorjahan JBasheer
[email protected]
BHS007-6–Assessment 1
Tutorial 1
MTTassay to evaluate the cytotoxic potential ofchemotherapeutic drugs in a cancer cell line
Practical week 821st–24thFeb 2023
1. Readthe“Assessment brief onBreo”
2. Read the “MTT assay to evaluate the cytotoxic potential ofchemotherapeutic drugs in a cancer cellline” protocol onBreo
3. Gothrough the “Tutorial1” slides
4. Go through the “various tasks/calculations” and the “Model PosterTemplate”
5. Practical sessions–timetable in the next slides
6. Attendance compulsory for all 3 practical sessions. Latecomers will not beadmitted for the practical sessions
7. Poster submissiondate10thMarch 2023 before 10am
Assessments
Assessment 1 (40%)–Practical posterpresentation
Inthis assessment you are required to submit a MSPowerpointposter based onthepractical sessions.
Poster Submission toBreo:
Friday, 10 March 2023: 10:00 am
Practical set up
Tuesday: (10am-5pm)–Room C320
Day 1:Part 1:Counting cells (Determine cell concentration/ml)
Day 1:Part 2: Plating the cells(In the FUMEHOOD)(Will be given pre-calculated cell count. You will use those cells to plate)
Wednesday (10am-5pm)
Day2:Part3: Treating cells withdrugs (In the FUME HOOD)
Friday (10am–5pm-Room C320) (Be prepared to stay longer!)
Day 3 :Part4: Terminating the experiment (In the FUME HOOD)
Week 8 (21st–25thFeb schedule forBHS007-6)
Wednesday: 22ndFeb: Please be prepared to stay longer.
Presentations: w11
Reagents used
Cell line used:A cancer cellline: B16F10 (Melanoma cell line)
Drugs used: two chemotherapeutic drugs
Drug Diluent: DMSO
Cell counting staining reagent:Trypanblue
Assay: MTT colorimetric assay
Helpful videos:
Counting Cells with aHaemocytometer: (6min)
https://www.youtube.com/watch?v=pP0xERLUhyc
Cell Viability and Cytotoxicity determination using MTTassay (5 min)
https://www.youtube.com/watch?v=8k8NtmpaP_U
MTTAssay for CellViability: (25 min)
https://www.youtube.com/watch?v=2mJ8lKfOgs0
Trypanblue (TB)
Vital stain permeable to cells depending on theirmembrane integrity
• Easy and inexpensive method
• Distinguishes live and dead cells
Under the microscope:
cells visualized blue–dead
cells visualized white/transparent–living
Commonly used ratio: 1part cells (ul):1part TB(ul)
The MTT cell proliferation assay is acolormetricassay
It measures the reduction ofyellow tetrazolium MTT(3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) by an enzyme,succinate
dehydrogenaseproduced in mitochondria.
When MTT is added to cells, it enters mitochondria where it isreduced to aninsoluble,coloured(dark purple)formazanproduct.The quantity
offormazanis presumablydirectly proportional to thenumber of viable cells)
This insoluble formazan product can be solubilized with an organicsolvent such as DMSO and the released, solubilized formazan reagentis
measured by spectrophotometer at570nm (OD reading)
MTT principle
MTT principle
Sincereduction ofMTT can only occur in metabolically active cellsthelevel of activity is ameasure of the viability of the cells.
Whencells die, they lose the ability to convert MTT intoformazan, thuscolorformation serves as a useful and convenient marker of only theviable
cells.
Inlayman’s terms,more purplecolorindicatesmore viable cells andmoreyellowcolorindicates, increased cell death
Task: Guess the cell viability
1 2 3 4
First take theaverage/meanof diplo and use that average in the below formula
Ateach drug concentration the cell survival is calculated by the formula:
Cell viability(%)=
(mean ODfrom cells + drugtreated wells ˗ mean ODfrom blankwells)
(meanOD from controlwells ˗ mean ODfrom blankwells)
Theconcentration of drug that is lethal to 50% of the cells(IC50) can becalculated from the dose-response curve made by plotting the cell survival
ateach drug concentration.
Or use formula (search yourself)
Calculating cell viability
*100
Experimental set up
| blank | Only cell growth media (no cells)(80ul+20ul = 100ul media) |
| control | Only cells (in cell growth media, but no drug) (80ul cells+20ul growth media) |
| C+D | Cells + drug (4 different concentrations) (80ul cells+20ul drug) |
| Drugs used | |
| Dacarbazineand Carboplatin |
| Dacarbazine | Carboplatin | |||||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | – | – | – | – | – | – | – | – | – | – | – | – |
| B | – | blank | blank | – | – | blank | blank | – | – | – | – | – |
| C | – | control | control | – | – | control | control | – | – | – | – | – |
| D | concn1 | C+D | C+D | – | – | C+D | C+D | – | – | – | – | – |
| E | concn2 | C+D | C+D | – | – | C+D | C+D | – | – | – | – | – |
| F | concn3 | C+D | C+D | – | – | C+D | C+D | – | – | – | – | – |
| G | concn4 | C+D | C+D | C+D | C+D | – | – | – | – | |||
| H | – | – | – | – | – | – | – | – | – | – | – | – |
Counting cells withHaemocytometer
• Device used for counting cells
• Cell density of cell line
• Contains 2 identical chambers
• Cell suspension is pipetted
• 9 large squares
• Count cells in 4 large corner
squares
Day 1:
Youwill be provided with a suspensionof a cancercell line
Add 20ulof the cell suspension to aneppendorftube.
Add 20uloftrypanblue to the cell suspension, mix well.
20µloftrypanblue-cell suspension is transferredto one of thechambers of the haemocytometer by carefully touchingthe coverslip at its edgewith the
pipette tip and allowing each chamber tofill by capillary action. The chamber should not be overfilled orunder filled.
Day 1: Part 1:Counting cells
Counting cells
Count clear and blue cells includingthese on the 2 boundaries of thesquare
• Calculate average from 4 squares
Add together the live and dead cellcount to obtain a total cell count
Divide the live cell count by the totalcell count to calculate the percentageviability.
Corner Count of viable
cells
Count of dead
cells
1 2 3
4
Trypan Blue is a “vital stain”; i.e. excluded from live cells.
Live cells appear colourless anddead cells stain blue.
•Calculation:-calculate the average from the 4 corner
squares.
1.Average viable (alive) cell count = Total number of viable
cells in 4 squares/4. Average = _________
2. 2.Average dead cell count = Total number of dead cells in
3. 4 squares/4. Average = _________
Cell concentration
Average viable (alive) cell count per square =Total number of viable cells in 4 squares / 4
Each large square of thehaemocytometer, withcover-slipinplace, represents a total volume of0.1 mm3
cm3is equivalent to approximately 1 ml
Multiply average by 104
• Dilution Factor = Total Volume (Volume
f cell suspension + Volume of trypan blue) /Volume of sample.
Final Concentration:times the dilution
factor by the cell concentration.
Primary square
Area=1.0mm2
Depth =0.1mm
Volume= 10-4ml
Trypanblue (TB)
Practice: Count the cells
Day 1: Part 2: Platingthecells
In the FUME HOOD!
Eg. Cell count (average): 50
Dilution factor: 50×2 = 100
Cell concentration present: 100x104cells or 1×106 or10,00,000 cells/ml
Required: 5x1040r50,000/96-wellisthe requiredconcentration
10,00,000 cells in 1000ul
? Cells in 1ul?
Ans: 1000 cells/ul
Ans: 50ul/well(1000 cellsx50 = 50,000 cells/well)
Todo
Cell counting:
See the protocol on BREO
Calculate cell concentration/ml
Calculation task: 1
Determinethe concentration of viable cells/ml in your sample
Whatis the percentage of viable cells in your sample?
Calculation task: 2
Calculatethe volume of this sample to dilute cell number to 0.5x106cells /ml where your final volume is 5ml of cells. (You will need4x104cells /well).
Youcan use c1v1=c2v2 formula
Experimental set up
| blank | Only cell growth media (no cells)(80ul+20ul = 100ul media) |
| control | Only cells (in cell growth media, but no drug) (80ul cells+20ul growth media) |
| C+D | Cells + drug (4 different concentrations) (80ul cells+20ul drug) |
| Drugs used | |
| Dacarbazineand Carboplatin |
| Daarbazine | Carboplatin | |||||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | – | – | – | – | – | – | – | – | – | – | – | – |
| B | – | blank | blank | – | – | blank | blank | – | – | – | – | – |
| C | – | control | control | – | – | control | control | – | – | – | – | – |
| D | concn1 | C+D | C+D | – | – | C+D | C+D | – | – | – | – | – |
| E | concn2 | C+D | C+D | – | – | C+D | C+D | – | – | – | – | – |
| F | concn3 | C+D | C+D | – | – | C+D | C+D | – | – | – | – | – |
| G | concn4 | C+D | C+D | C+D | C+D | – | – | – | – | |||
| H | – | – | – | – | – | – | – | – | – | – | – | – |
(See calculations next slide: Prepare 5ml of drug. Add 20ul/well fromthere)
Add required amount ofMedia only(20ul) to the‘blankcells’
Add required amount ofgrowth media (20ul) to the ‘control cells’
Add required amount of‘drug 1’or‘drug 2’to the ‘treatedcells(C+D)’ in the fume hood (cell culture)
Leave the plate overnight at370C
Day2–Part 3: Treating cells with drugs
In the FUME HOOD!
Below are the concentrations required for both the drugs. Use the above stock to calculatethe required concentration.
| Drug | Stock (uM) | Required volume (ml) |
| Drug1 | 500 | 5 |
| Drug2 | 750 | 5 |
| Required Concentration(uM) | Stock volume (ul) | Diluent (DMSO)volume (ul) |
| Concentration 1 | 0.01 | |
| Concentration 2 | 0.1 | |
| Concentration 3 | 1 | |
| Concentration 4 | 5 |
Use the formula: C1V1-C2V2
C1V1=C2V2 is used to calculate an unknown quantity where two solutions/mixtures areproportional …
(https://www.mathcentre.ac.uk/resources/uploaded/c1v1c2v2.pdf)
C1V1 = Concentration/amount (start) and Volume (start)
C2V2 = Concentration/amount (final) and Volume (final)
Calculation task:3
Calculate the drug solutions. This will become your working stock.
MTT working solution: (5mg/ml stock).MTTpowder is dissolved inPBS, filter sterilized and covered in aluminum foil to avoid light.
———————————————————————————————
Add20µl of MTTto each well.shake each plate for 5 minutes on aplateshaker
Incubate at 37°C in the dark for ~2hrs(this time depends oncelldensity and cell type).
Add 100µl DMSO to each well.
Mix the cells well to dissolve formazan crystals.Incubate the plateat room temperature for 15 minutes.
Measure absorbance at 570nm
Day3-Part4: Terminating the experiment
In the FUME HOOD!
Example
| Drug | Cell viaiblity (%) | |
| Concn(uM) | Dacarbazine | Carboplatin |
| 0.05 | 69 | 85 |
| 0.5 | 41.78 | 62 |
| 1 | 11.92 | 35 |
| 5 | 2.01 | 21 |
To do–Part 4
Calculation:task4
DeterminationofcellviabilityandIC50
1. Determinethecellviabilityateachdrugconcentrationbytheequation:
2. PlotdatainexceltodemonstratetheeffectofeachconcentrationofdrugsonthecellsanddeterminetheIC50.
Some questions which you could answerin yourposter:
Explain the role of trypan blue in this experiment
Explain the role ofdrugs usedin this experiment
Discuss MTT assay
Determine the cell concentration and % viable cells based on cellcounting
Whatis the OD of yourblank (media only)?Why?
Discuss the effect ofDacarbazineand Carboplatintreatmenton cellviability. Which is more effective in cancer therapy?
Calculate the IC50 of above drugs
Compare your data with published literature. Is it similar or contradictory?
Imp: Discuss one figure of cell viability experiment from the literature
Title and author:
Includea conciseposter title, your name, student ID number, unit code, assessmentnumber andaffiliation(See poster template put onBreo)
Introduction:
Introduce the topic with an outline including background information frompublished literature and relevance.
Aim:Clearlystate thescientificaim of the studies.
Methods:
Summarisein brief the various experimental steps, in the form of a methods sectionin a journal article, perhaps with the aid of a flow diagram.
They should be written in the past tense and in paragraphs (no bullet points). Theyshould contain sufficient detail to allow someone else to reproduce
yourexperiments, but avoid unnecessary detail. No need to mention the materials.
Some tips in preparing the poster
Most imp: Read Assessment brief carefully
Results/Data Analysis
You must begin your results section with a paragraph to briefly recap the purpose ofthe experiment and state what data you will present in thissection
Data should be displayed in figures or tables that are easy to read and clearlylabelled. Results should be described in the accompanying text and
the relevantfigures/tables should be referred (in-text citation) to where appropriate. Allfigures and tables should be separately numbered and should
have a figurelegend explaining the contents of the figure.
Createprofessional quality graphs/tables using Excel or similar program (no handdrawn figures please).
Donot interpret the meaning of findings in this section.
Discussionand conclusion:
Explainthe data andsummarisethe main outcomes with respect to achieving thestated aim.
Reference list
(Not more than 5)
This (and the in-text citations) must be formatted according to the UoB Harvardstyle, available here:
https://citethemrightonline.com/
Important:
A cell viability figure from the published literature (provide reference).You willneed to pick a figure from published literature where the researchers have
showedmeasurement of cell viability after the drug treatment in a cell line/primary cells.
Example:Acell viability figure from thepublished literature
(provide reference).
Sadeghi-Aliabadiet al., 2010
YouMUSTNOTcopyanytextfromanysources,evenifyoucitethesource–youmustwriteaboutwhatyouhavereadinyourownwords.-thisisveryimportant.
Studentsareremindedoftheirresponsibilitiesconcerningacademicintegrityandthatplagiarism(theuseofothers’words,publishedorunpublished,andfailingtoacknow
Thisisanindividualassessment,socollusionisalsoanacademicoffenceincludingthehelpfromanyexternalsource.
AcademicIntegrity
Thank You