MTT assay

One well can contain 200l of solution.

Label plates with cell type, drug, date (24, 48 72 hours), name

For cancer cells the readings have to be more than 1

For trouble shooting refer: (attc mtt assay, 2007)

Steps:
Prewarm the media and serum
Put in 25l media into Petri dish
Prepare compounds. For extracted compounds, we need the final concentration be
100g/ml. As it will be diluted when put into well, we prepare 200g/ml of stock.
The original stock concentration is 10mg/ml. 1 well contains 100l. For 1
compound needs to be filled in 6 wells in 1 plate (A and B). so =600l=1ml.[2ml]
50000X=2001
X=0.004ml
=4l (this is the amount of original stock needed). Or else we can put in 20l
and top up to 5ml.
To make it 1ml, we add in 980l of DMEM.
The negative control will be at the H lane, with cell and media only (IC100). The
3 lines at the right hand side will be used for positive control.
Put 100l of the compounds into wells A and B respectively and serial dilution
(100l) from B to G. Discard.
So total volume in the treatment well is 200l.
Detach cells
We need 10 ml (1 plates)of cells with 106 cells (so each well has 10000 cells).
Take out 10l of cells.
Mix with 10l trypan blue. One small square of the hemocytometer has 100nl
space. (Hemocytometer - Wikipedia, the free encyclopedia, 2007. There should
be 10 cells
Take out the correct amount of cells. Top up to 10000l.
Put in 100l of cells in every seeding well (from A to H).
Incubate for 24 hours
Discard the media in the seeding wells (100l)
Transfer the solution in treatment well to the seeding well
Incubate for 72 hours
Discard 170l of the solution
Put in 20l of MTT (2ml per plate).
Incubate for 4 hours, 37C
add 100l of DMSO (solvent, MTT has become formazan, crystal dissolve it for
ELIZA). About 10ml per plate. [no need]
Incubate for 1hour, mixing with multichannel pipette (avoid bubbles) [no need]
 Read with ELIZA, wavelength=570nm and 650nm. Od570-0d630= sample signal.
0d630=reference.
Read for the second time (to compare, maybe got bubbles or low/high readings at
a particular part)

(mtt assay, 2007)

IC50 Determination by the Standard Curve Method

This is to get a standard curve for the generation of IC50.

A standard curve was prepared for each set of experiments using the same
solution of cells. A microplate containing 40 x 103 cells/ well was prepared. Then,
five dilutions (20, 10, 5, 2.5 and 1.25 [ x 103cells/well]) were prepared from the
original 40 x 103 cells/well. Cells were seeded by transferring the cell dilutions
into the appropriate well of the microplate.

To determine the IC50 value using the standard curve method, a reading of the
absorbance representing 104 cells (50% of the total population in each well) was
specified. This absorbance value was then used to determine the IC50 value using
the X-axis intercept of the dose-response curve, as described in the conventional
method above.

(CellTiter96 Aqueous Non-Radioactive Assay, 2007)