MATERIALS & METHODS
MATERIALS & METHODS
Materials and Equipment
1) Complete
media (DMEM) suitable for chosen cell type (adherent cell)
2) 96-well
tissue culture plates
3) Inverted
microscope
4) ELISA-type
spectrophotometer
5) Micropipette
(200 ml) and sterile yellow pipette tips
6) Test agent: Panadol
1) Neutral
red (from stock solution of 4mg/ml): Diluted 1:100 into medium (DMEM), incubated overnight
at 37oC and centrifuged before use.
2) 1%
CaCl2 / 0.5% formaldehyde: 5g of CaCl2.H2O was weighted
and dissolved in 50ml of distilled water making up the 10% CaCl2. 6.5ml of 37%
formaldehyde was mixed with 50ml of 10% CaCl2 and 445ml of distilled
water.
3) 1%
acetic acid / 50% ethanol: 4.75ml of acetic acid was mixed with 250ml of 95% ethanol and
245ml of distilled water.
1) Cells
of actively growing culture were resuspended by standard trypsinization procedure.
2) Cells
were counted and appropriate number suspended in medium was accurately allocated, whereby
~80% confluence can be achieved at time of addition of test agent.
Cell Count
Average number of viable cells = (3 + 5
+ 7 + 4 + 3)/5 = 4.4
4.4 *104 * 2 = 8.8 * 104 cells/ml
In 10ml of medium,
Total cell count = 8.8 * 105 cells
8.8 * 104 cells --------- 1ml
X
cells
--------- 0.2 ml
X = 8.8 * 104 * (0.2/1) =
1.76 * 104 cells per well
% Viability = 100%, no dead cells observed.
Flow Diagram of Procedure for Neutral Red Assay
The
percentage cell population viability was calculated using the following formula: -
% Viability = Mean absorbance of experimental cells / Mean absorbance of control cells
The following is a diagrammatic representation of the 96- well plate filled with graded dilutions of test agent: -
Key:
= 150 mg of paracetamol in 150 ml of DMEM
= 100mg of paracetamol/100ml of DMEM
S/no. |
Color Code |
Test Agent |
1)
|
|
Positive
control 200 ml DMEM |
2)
|
|
Negative
control 200 ml of 70% ethanol |
3)
|
|
Test 1: 2.0mg
of paracetamol in 0.2 ml of media 200 ml of test medium |
4)
|
|
Test 2: 1.6mg
of paracetamol in 0.2 ml of media 160 ml of test medium + 40ml of DMEM |
5)
|
|
Test 3: 1.2mg
of paracetamol in 0.2 ml of media 120 ml of test medium + 80 ml of DMEM |
6)
|
|
Test 4: 1.0mg
of paracetamol in 0.2 ml of media 100 ml of test medium + 100 ml of DMEM |
7)
|
|
Test 5: 0.8mg
of paracetamol in 0.2 ml of media 80 ml of test medium + 120 ml of DMEM |
8)
|
|
Test 6: 0.6mg
of paracetamol in 0.2 ml of media 60 ml of test medium + 140 ml of DMEM |
9)
|
|
Test 7: 0.4mg
of paracetamol in 0.2 ml of media 40 ml of test medium + 160 ml of DMEM |
10) |
|
Test 8: 0.2mg
of paracetamol in 0.2 ml of media 20 ml of test medium + 180 ml of DMEM |
