Description

Procedure in Brief

:

Cells were started at a low density, so that we could follow the increase in cell number over time. Flasks were seeded with equal numbers of cells in each flask, but with an approximately equal number of cells in each flask. The flasks were incubated overnight to allow the cells to adhere to the flask surfaces. The cells were then randomly divided into three groups and the medium was replaced with medium containing either 5, 10, or 15% (this was Day 0).

1. Setup of flasks:

For this method, two confluent flasks of cells were used. The cells were each split 1:8 for a total of 16 flasks.

– The medium was removed from the 2 flasks and discarded.

– The flasks were washed with 6ml each of phosphate buffered saline (PBS) and

discarded.

– Aliquots of 2.0ml trypsin-EDTA were added to each flask, and incubated for

approximately 8minutes.

– Fresh medium (6ml) was added to each flask and the cells were pipetted up and down

6 times to break up the clumps of cells.

– The two cell suspensions were combined in one 50ml conical centrifuge to create one cell suspension.

– Aliquots of 1.0ml were placed into each of the 16 flasks along with 5ml of fresh

medium (10 %serum)and allowed to incubate overnight.

2. Day 0: Initial Cell Counts:

Three of the flasks were chosen at random and the cells were counted by standard methods. Three flasks were used at this step to determine the initial cell seeding number. By measure the number of cells in three flasks, we will get an average value to use as a starting point, and, by calculating the standard deviation, we will get a measure of the variability of the cell numbers between flasks.

– Cells were trypsinized by the procedure described under Item #1.

– Following trypsinizaton, the 2ml of trypsin was diluted with 4ml of fresh medium to

create the cell suspension.

– The three samples were spun at 1000rpm for 5 minutes to create a cell pellet and

concentrate the cells.

– Most of the medium was removed (aspirated) and discarded. Approximate 0.5 ml of

medium was left in the tube so the pellet would not be disturbed during the

aspiration process.

– The cell pellet was resuspended with a P-1000 micropipet, the volume was measured

accurately, and then they were stored on ice until counted.

Cell Counting

– The cell suspension was mixed immediately be sampling for counting. An aliquot of

50ÃŽÂ¼l of cell suspension was added to a microcentrifuge containing 50 l of trypan

blue and mixed gently.

– A glass Pasteur pipet was used to suck up a sample of this mixture by capillary action.

– A haeomocytometer was loaded on both sides with the sample.

– Under a microscope, the cells were counted in the 4 corner quadrants of the grid as

well as the center square of the grid (total of 5 boxes per side of the

haemocytometer).

– The number of unstained cells (viable cells), and the number of blue cells (non-viable

cells) were counted separately. The total number of cells was obtained by

adding these together.

3) Treatment of Flasks Starting on Day 0:

– Twelve of the remaining flasks were divided into 3 groups of 4 flasks. (One flask was

not used.).

– The medium for each of the groups of 4 flasks was aspirated and replaced with 6ml of

either 5, 10, or 15% serum-containing medium and placed back in the incubator.

– On each day the flasks were sampled, 1 flask from each group was used for cell

counting, and the remaining flasks had their medium replaced with fresh

medium of the appropriate serum concentration.

4) Data Analysis:

– For each cell count, the total number of cells were added up for the 10 quadrants that

were counted.

– Viability calculation was carried out by standard practice. The total number of cells

was calculated by adding the number of non-stained cells plus the blue-stained

cells. Then the number of non-stained cells was divided by the total number of

cells and the value was multiplied by 100 to determine the viability in units of

percentage (%).

– The total number of cells was determined by adding up all of the cells (viable and non-

viable) from all 10 quadrants.

– This value was divided by the volume for 10 boxes (0.001ml).

– The cell concentration was multiplied by 2 to correct for the dilution with trypan blue.

– The cell concentration was then multiplied by the total volume of cells to determine

the total number of cells in the flask.

– For the three Day 0 samples, the average cell number was calculated as well as the

standard deviation.

– The data were graphed with the Ã¢â‚¬Å“Time (Days)Ã¢â‚¬Â on the x-axis, and the total cell number

in each flask on the y-axis.

5) Interpret the results:

– The trend in the cell counts over time were summarized and the key values were

explained and a conclusion was reached.

Assignment

:

1) Summarize the data as indicated above.

2) Create Ã¢â‚¬Å“Figure 1Ã¢â‚¬Â as described above.

3) Write a results section that explains the trends and key values that relate to the hypothesis.

4) Write a paragraph that states the conclusion and supports that conclusion. (Note: if you cannot reach a firm conclusion, state that, and explain why you cannot. Explain what you think you would need to do to repeat the experiment to get a more conclusive result.)