Cell Viability / Cytotoxicity assay

(MTT assay)
 Based on
Trypan blue
membrane
staining
integrity

Diacetyl
Dye uptake assay
fluorescein

Labeled
chromium uptake 51Cr method
Cell Viability / assay
Cytotoxicity
assay Enzyme release
LDH
assays

Based on
Annexin V
apoptosis

Colorimetric
MTT assay
assay 2
 Based on
Trypan blue
membrane
staining
integrity
 Measurement of cell viability and

Diacetyl cytotoxicity at short-time.

Dye uptake assay
fluorescein  Identify the dead/live cells at the
time of assay.
Labeled  Many times, when the cells are
chromium uptake 51Cr method
Cell Viability / assay subjected to drugs, the effects are
Cytotoxicity not immediate, but may be
assay Enzyme release
LDH observed after several hours or
assays
days.

Based on
Annexin V
apoptosis

Colorimetric
MTT assay
assay 3
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay

• A colorimetric assay for assessing cell metabolic activity.

• For determining mitochondrial dehydrogenase activities in the living cells.

• NADH reducing the tetrazolium dye MTT to its insoluble formazan, which has a purple color.

• Measurement of MTT-formazan in an ELISA plate reader.

• The linear relationship between cell number and signal produced is established.

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 MTT and related tetrazolium salts

MTT XTT MTS

(3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium
bromide)
(3-(4,5-dimethylthiazol-2-yl)-
WSTs
(2,3-bis-(2-methoxy-4-nitro-5-
5-(3-carboxymethoxyphenyl)- (water-soluble tetrazolium
sulfophenyl)-2H-tetrazolium-
2-(4-sulfophenyl)-2H- salts)
5-carboxanilide)
tetrazolium)

Insoluble
Water soluble Water soluble Water soluble
purple formazan product product product
product

Absorbance 550- Absorbance

Higher sensitivity
720 nm maximum at 490
nm

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 Application

• For assessing cell viability.

• To measure cytotoxicity (loss of viable cells).

• Drug screening on cell lines and/or patient samples (IC50).

6
 Materials and Equipments

96 well 37ċ CO2

Samples ELISA Reader
microplate incubator

DMSO (Dimethyl Multi-channel Cell culture

MTT powder
Sulfoxide) pipette media

Inverted
microscope

MTT is toxic and harmful. MTT is light sensitive, hence protect from light. 7
 Optimization of Assay Condition
Determination of the optimal cell count and incubation period for cell line:

1. Harvest suspension cells by centrifugation. Adherent cells should be released from their substrate by trypsinization or scraping.

2. Prepare serial dilutions of cells in culture medium from 2.5 x 10 ⁴ , 1.25 x 10 ⁴ , 6.2 x 10 ³ ….0 cells per well.

3. Incubate the cells under conditions appropriate for the cell line for 6 to 48 hours.

4. Add 10 μL of MTT Reagent to each well.

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 Optimization of Assay Condition

5. Return plate to cell culture incubator for 2 to 4 hours.

6. When the purple precipitate is clearly visible under the microscope add 100 μL of Detergent Reagent to all wells.

7. Leave plate with cover in the dark for 2 to 4 hours or overnight at room temperature.

8. Measure an absorbance on a microplate reader (550 - 720 nm).

9. Establish the standard curve by plotting the number of cells on the x-axis and the absorbance on the y-axis.
10. The number of cells within the linear portion of the plot and yield an absorbance of 0.75 - 1.25 is suitable.

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 Precautions

For floating type cells, please use a V bottom plate.

If the time from starting incubation to taking a measurement is over 48 hrs, it is necessary
to exchange the media.

Tilt the plate when removing the media to avoid touching the cells with the tip of the
pipette.

For floating type cells, centrifuge a V bottom plate with a microplate rotor, and then
remove the media after the cells settle out of the solution with care not to suck in cells.

The exposure time depends on the test substance and purpose of the experiment. If the
substance is highly toxic to the cell, short exposure time will be appropriate. If the
substance slowly affects cell function, longer exposure time may be appropriate.

10
 Procedure

Cell culture in 96 well Incubation for 24-48 hrs in Treatment of Incubation for 6, 12, 24,
plate (10x10 ⁴ cells/well) a CO2 incubator drug/compound 48 hrs in a CO2 incubator

Measure an absorbance Solutions (DMSO or methanol Incubation for 3-4 hrs in a 10 μg of MTT
(550 - 720 nm) or ethanol) is added for 30 min CO2 incubator (5mg/ml) reagent is
in dark room added and mixed
11 well
Procedure

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 Calculating the Cell Survival Rate

1. Survival rate (%) =

2. Survival rate (%) =

Example:
Background absorbance: 300
Sample A: 700
Control: 800
1. Relative Ratio (Sample/Control) : 700 / 800 = 87.5%
2. True relative ratio: (Sample-Background)/(Control-Background) :
(700-300)/(800-300)=80%

A: Absorbance
b (blank/background): no cell, as a background control 13
Control: healthy cells with 100% viability, no drug
 IC50 in MTT assay

 Determination of IC50 values:

 Different dose ranges are required.

 After measurement by plate Reader .

 The MTT assay is suitable for the measurement of drug sensitivity in established cell lines.

For cell lines: the decrease in cell number reflects cell growth inhibition and the drug sensitivity is then usually specified as the

concentration of the drug that is required to achieve 50% growth inhibition as compared to the growth of the untreated control

(50% inhibitory concentration, IC50).

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 IC 50 calculating

1.
• Excel

2.
• Graph pad prism software

3.
• Online tool (https://www.aatbio.com/tools/ic50-calculator)

15
1. Excel IC 50 calculating

Survival rate (%) =

16
1. Excel IC 50 calculating

110
IC50
100
f(x) = − 0.149536680480679 x + 100.906468136087
90

80
Cell viability

70

60

50

40

30

20

10

0
0 100 200 300 400 500 600 700 800

Concentration

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Y = mx + C → (y = -0.1495x + 100.91)
Y = % Inhibition
x= Concentration
C = Constant
m= Coefficient

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2. Graph pad prism software IC 50 calculating

19
20
21
22
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 IC 50 calculating
3. Online tool (https://www.aatbio.com/tools/ic50-calculator)

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 Troubleshooting

Problem Possible cause Solution

1. The absorbance Too many cells per well. Decrease cell density at plating.
reading exceeds the
Contamination of culture with bacteria or yeast. Discard.
upper limit of the
machine Too long of an incubation time. Shorten the incubation time.
Cell number per well is too low. Increase cell density at plating.
Incubation time for reduction of MTT is too short. No Increase incubation time with MTT
purple color visible in cells when viewed under Reagent until purple color is evident.
microscope. Longer incubation of up to 24 hours
may be required.
Incubation time for solubilization of formazan dye too Increase incubation time with
2. Absorbance readings short. Detergent Reagent or incubate at 37°C.
are too low View under microscope to ensure no
crystals remain out of solution.

Cells not proliferating due to improper culture conditions Check that culture conditions
or inadequate time of recovery after plating. (medium, temperature, humidity, CO2,
etc.) are appropriate.

27

Problem
3. Blanks (medium only) give high
absorbance readings
4. MTT Reagent is blue-green
5. There is a high variation in the
data
6. No color or less color
development even though the
number of cells seems to have
increased
Troubleshooting
Possible cause
The medium is contaminated with
cells/bacteria/yeast
Contamination with a reducing
agent/bacterial contamination
Excessive exposure to light
Inaccurate pipetting
There are bubbles on the surface of
the media
Cell viability of each cell has been
lowered because of too many cells
Solution
Discard. Check medium before
plating. Use sterile technique for cell
plating in biological hood
Discard. Remove aliquots of new
MTT Reagent using sterile technique
Store solution in the dark at 4°C
Check accuracy of pipette
Remove the bubbles using a pipet tip
Reduce the number of cells
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Thank
you
29