Roughnotes 2 new:Protocols:Cell culture protocols:Cell titer vs. CC test
January 21, 1993
Cell Attachment Assay
(Promega Cell Titer Method vs. Coulter Counter)
Objective:
To correlate absorbance readings at 550 nm (on the plate reader) to cell
number, which will be confirmed by Coulter counter results; accuracy of using
the plate reader will be determined.
1) 1 surface (to eliminate surface dependence) -- 96-well tissue culture plate
2) w/ serum condition
3) variation of cell # (in triplicate) -- 102, 103, 104, 105 cells/well
Materials needed:
medium w/ serum
sterile 96-well flat bottom tissue culture plates (2)
polyprop tubes (50 and 15 ml)
repipettor
sterile repipettor tubes (50 µl, 100 µl, 15 µl)
aspirator
Coulter counter
Coulter counter tubes (27)
filtered ISOTON
trypsin
pipetman (P200, P1000) and tips
glutaraldehyde
methanol
non-sterile PBS
sterile PBS
dye solution
solubilization soln
container (w/ moist paper towel)
plate reader
PLATING CELLS
1.
Add 50 µl media w/ serum to proper wells in the 2 tc plates with repipettor.
(see attached data sheet for this experiment)
w/s: 50 µl x 4 rows x 3 cols x 2 plates = 1.2 ml —> need 2 ml.
2.
3.
Place plates in incubator; note time on data sheet.
Prepare cells (choose enough plates):
a) Take plates out of incubator; aspirate off medium; and wash plates
(as usual) with 2 ml trypsin.
b) Trypsinize cells with 3 ml per dish.
c) Stop action with 7 ml DMEM medium w/ serum.
d) Centrifuge down (important: combine plates into one tube).
Roughnotes 2 new:Protocols:Cell culture protocols:Cell titer vs. CC test
January 21, 1993
This is done by using the medium with cells from one plate to wash
off
the cells on another plate.
e) Aspirate, leaving pellet in place.
f) Add 1 ml DMEM w/ serum w/ pipetman.
50l x 6 samples = 0.3 ml for Coulter counting 50 l of 105 cells
50l x 3 samples = 0.15 ml for Coulter counting total# cells
Total = 0.45 ml ====> 1 ml medium needed
g) Count cells with Coulter counter (bring a Pipetman to mix well)
-- need 9.95 ml filtered ISOTON + 50 l cell suspension per
Coulter counter tube
-- (# cells counted) / (0.5 ml vol.) x 10 ml = total # cells in 50 l
(count 3 samples -- need 150 l cell suspension)
-- (total # cells in 50l) / 0.05 ml = total cells/ml
-- cell suspension remaining = 1.0 ml - 0.15 ml = 0.85 ml
-- total remaining cells = total cells/ml x 0.85 ml
-- want: total remaining cells/X ml = 105 cells / 0.05 ml
Volume to add to total remaining cells = X ml - 0.85 ml
h) Do serial dilutions to get 104, 103, 102 cell concentrations.
4.
5.
6.
7.
8.
9.
10.
11.
Add 50 µl of each concentration of cells (102, 103, 104, 105) in triplicate
to the appropriate wells. Shake around plates to make sure that cells
are well-distributed.
Incubate for 4 hrs. Note on data sheet time plated. (Thaw dye solution)
After 4 hrs, wash plate for absorbance reading w/ sterile PBS 4x (100l)
all wells.
Add 15 µl of dye. Caution! Irritant. Avoid contact with skin or eyes!!
{ 15 µl x 4 rows x 3 cols = 0.15 ml —> 0.2 ml)
Incubate dye-filled plates for 4 hrs. Note time on data sheet.
After 4 hrs, add 100 µl solubilization soln to each well in the fume hood.
Waste should go in organic waste container.
{ 100 µl x 4 rows x 3 cols = 1.2 ml—> 2 ml)
Seal and put into container with moist paper towel overnight.
Also, after 4 hours, on plate for Coulter counting:
a) 100 l cell suspension + 200 l from 2 PBS washes per well
= total 300 l floating/dead cells + 9.7 ml ISOTON per tube (12)
Roughnotes 2 new:Protocols:Cell culture protocols:Cell titer vs. CC test
January 21, 1993
b) Add 100 ml trypsin per well to trypsinize attached cells.
Remove 100 ml 'attached' cells + 9.9 ml ISOTON per tube (12)
c) Coulter count all tubes.
12.
Next morning, read absorbance at 570 nm wavelength with 630 nm
reference, from plate for absorbance reading.
PLATE READER
Note: Instructions of keys to punch are in boldface type.
To check date and time:
1.
2nd TIME
2.
Date: (If date needs to be changed, press EDIT, if not, press ENTER and
go to step 5)
3.
Input actual date: e.g. 012193
4.
12/24 h (1/2): 2 (international 24 hr mode)
5.
Time: (If time needs to be changed, press EDIT, if not, press ENTER)
6.
Enter in time: e.g. 930 ENTER
READY 9:30 (Standby mode)
To program:
This program will have the reference wavelength.
1.
2nd PROG or 2nd EDIT
2.
Test Nr: 5
3.
Test Name: enter
4.
dual wavelength: y
5.
Meas. Filter (1-6): 3 enter (550 nm)
6.
Ref. Filter (1-6): 1 enter (620 nm)
7.
Result (1-7): 5 enter (triplicates)
READY 9:30 (Standby mode)
This program will not have the reference wavelength.
1.
2.
3.
4.
5.
6.
7.
8.
2nd PROG or 2nd EDIT
Test Nr: 3
Test Name: enter
dual wavelength: n
Meas. Filter (1-6): 3 enter (550 nm)
Result (1-7): 7 enter (triplicates)
options (y/n): y
blankmode (1-4): 1 enter (average of col 1)
Roughnotes 2 new:Protocols:Cell culture protocols:Cell titer vs. CC test
9.
print format (1/2)?: 1 enter (vertical)
10.
print limit (y/n)?: n
READY 9:30 (Standby mode)
To run program:
(see attached pages 4-3 and 4-4 from the manual.)
January 21, 1993