Cell Culture
Counting Cells in a Hemacytometer
1. Clean the chamber and the cover slip.
2. Put the cover slip above the chamber
3. Add around 10µl cells. D o not overfill
4. Place the chamber in the inverted microscope under a 10X objective. Use
phase contrast to distinguish the cells.
5. Count the cells that are in the large gridded square (1mm2). The gridded
square is circled in the graphic. Multiply by 104 to estimate the number of cells
per ml. Prepare the same samples and average the amount of cells that we
obtain.
Freezing Cells
1. Prepare freezing medium and store at 2-8oC until we use it. Different cell lines
might need different type or amount of freezing medium.
2. For adherent cells, detach the cells from tissue culture vessel. Resuspend the
cells in complete medium that fit that cell type.
3. Determine the total number of cells and percent viability using a
hemacytometer
,
cell
counter and Trypan Blue
exclusion
,
or
the Countess®
Automated Cell
Counter
.
According
to the desired viable cell
density
,
calculate
the required volume
of
freezing
medium
.
4. Centrifuge the cell suspension for 5-10 minutes at 100 – 200 x g. Discard the
supernatant.
5. Resuspend the pellet in cold freezing medium at the recommended viable cell
density for the specific cell type.
6. Dispense the cell suspension into cryogenic storage vials. Aliquot them
frequently and gently mix the cells to maintain a homogeneous cell
suspension.
7. Freeze the cells in a controlled rate freezing apparatus. Decreasing the
temperature 1oC every minute. Put the cryovials with the cells in an
isopropanol chamber and store at -80oC overnight.
