#6 Cell Viability Assay Using Cell Counting Kit 8 (CCK8)—Better than MTT, XTT

Before the experiment, grow enough cells to use (one 10-cm dish
usually enough) and make sure they are healthy and actively growing
and not "static" culture (culture that has been confluent for days).

**Day 1:
Trypsinization procedure:
a. Remove media from 10-cm dish (aspirate) using pasteur glass pipette
with a sterile yellow tip at the end.
b. wash gently with 1xPBS 1.5ml (cross motion once/twice), switch the
yellow tip at the end of Pasteur to a clean one, then aspirate 1xPBS
off.
c. Add trypsin-EDTA solution (for 10cm, 1.5ml)  incubate at 37C CO2
incubator for 15min
The trypsin digests the receptor attachments of cells to the dish and
"lifts" the cells off the plate
d. Check if cells have lifted by looking under the microscope. Cells
should be rounded and "float"
around. If most are detached from the bottom of the dish, place plate in TC hood
e. Add 5ml complete media (the cells' preferred media containing
10%FBS/1%PS). Check the media list to make sure to give correct media.
  The FBS in the media inactivates the trypsin.
f. Measure cell number in the sample.  First, make a 1:10 dilution in
a microfuge tube= 100ul cells + 900ul 1xPBS.  Then, from this
dilution, take 100ul diluted cells + 100ul Trypan blue
The trypan blue penetrates dead cells, making them appear blue under the scope.
g. Mix the trypan blue/cell solution, take 10ul and pipette carefully
into a hemacytometer chamber. Count only the viable (clear) cells.
Blue cells are dead.
h. The formula that will give you the number of viable cells will be:
Dilution factor x average of 4 quadrants counted x 10^4 (hemacytometer
volume conversion/mL)
For example =   1st dilution was "10" x 2nd  dilution was " 2" x "45"
average quadrants x 10^4 =
900x10^4 cells/mL or 9x10^6 cells/mL
i.Now you have  a known concentration of cells to use in the experiment

Seeding the Experiment in 96 well plate
a.      Calculate how many wells you need. Make 2-4 extra.
b.      You need usually 1.5-3 x 10^3 cells per 96 well. The cells need to
be in a final volume of 100ul because the wells are very small.
c.      For each experimental or control group you need triplicate samples.
d.      Prepare appropriate amount of cell solution and dispense carefully
100ul per well (avoid outer wells of the plate as they tend to
evaporate the media more easily)
e.      Place plate at 37C incubator CO2. Cells will attach overnight

DAY 2;
Experimental treatment day
a.      You usually have an experimental treatment on day 2. Either it is a
virus infection (Adenovirus) or a transfection (DNA plasmid).  Also,
some cells will be treated with a drug, etc.
b.      Prepare a master mix of your "agent" and distribute 10ul per treated well.

DAY3
#day1 CCK8 cell viability assay and A450 reading.
a.      Add 10ul of CCK8 reagent to each well carefully not to introduce any bubbles!
b.      Place plate back at 37C incubator for 1-2 hours. 2 hours best.
c.      Take plate out and read at A450 with plate reader  save results or print.
d.      Bring plate back to TC hood, carefully aspirate the media/cck8
mixture off. CCK8 is not a toxic reagent, but it's best to remove it
e.      Place carefully 100ul complete media (FBS/PS) on the wells  plate
back into incubator
(if the cells need to have androgen or estrogen for the experiment,
the media should contain this).

Day 5: 2 days later
#day3 CCK8 assay reading

Day 7: 2 days later
#day5 CCK8 assay reading

Day 9: 2 days later
#day7 CCK8 assay reading

The experiment will give you a viability curve for day 1, 3, 5, and 7
of CCK8 assay. The A450 will increase with each day's growth. If
antigrowth treatments are applied to some groups, those cells of
course will not grow as much as control and CCK8 assay color will be
lighter.
The higher the A450, the more cells in the well. The lower the A450,
suggests cell loss or death.