BENG162 Memo

To: Dr. Melissa Micou

From: Group 7 Members Michael Zhao, Erin Kizer, and Nicolas Etcheverry
CC: Teaching Assistants Ryan, Andrew, Jared, and Gabriela
Date: 23 October 2012

Re: Encouraging Results of Millipores Scepter Technology

Background / Introduction
The procedural elements highlighted by this experiment helped to provide a comparison between two different cell counting
methods. The first method employed the use of a hemacytometer to establish the cell count as well as cell viability using trypan blue
staining, and the second method utilized the Scepter instrument to count cells via voltage/resistance increases. Cell densities were
recorded for both methods, and the data from different groups were pooled together to obtain a better understanding and comparison
of both cell counting techniques.
Experimental Objective
The purpose of this experiment is to determine the cell density of a mammalian cell suspension with both a hemocytometer and a
Scepter to determine how well correlated they are.
Summary of Results
Three samples were counted with the hemocytometer because the first two sample counts varied by more than 10%. Two counts per
trial for a total of six counts over three trials were recorded. The counts are shown in Table 1. The average live cell count was
determined to be 170 20 cells; the average dead cell count was determined to be 29 11 cells; the total cell count was determined to
be 200 30 cells. Trypan blue was mixed with the suspension samples to mark dead cells. Taking the trypan blue dilution into
consideration, the following formula was used to calculate cell density of the original cell suspension:
Cell Density (cells/mL) = 10,000 x 1.25 x N, where N = cell count
The total cell density in the mammalian suspension determined by the hemocytometer was found to be 2.5 x 106 cells/mL. However,
cell viability ([live cells / total cells] x 100) in the suspension was calculated to be 86%. Therefore, live cell density in the suspension
was found to be lower with a calculated value of 2.1 x 10 6 cells/mL. The total cell count for the original 0.5 mL cell suspension was
calculated to be 1.1 x 106 cells.
For our Scepter cell count, we used a 60 m sensor to test a 1:10 dilution of a sample of the same mammalian cell suspension. The
cell density of the diluted sample was determined to be 1.3 x 10 5 cells/mL, and the cell density of the original suspension followed to
be 1.3 x 106 cells/mL. The total cell count for the original cell suspension based off the Scepter results was 7.5 x 10 5 cells. Our results
were obtained by setting the low gate at 6.00 m and the
Counter
Cell
Sample 1
Sample 2
Sample
high gate at 24.2 m. Average cell diameter was given by
Individual
Condition
3
the Scepter to be 15.3 m. Also, graphical data on the
Live
160
194
153
1
Scepter screen pointed towards a size distribution of cells
Dead
42
25
17
between 12 and 19 m indicating that the majority of the
Live
162
204
148
2
cells were within this diameter range.
Dead
41
29
17
Table 1 Cell counts with the hemocytometer.
Class data from both the hemocytometer and the Scepter
were pooled together and plotted. The correlation between the cell densities given by the two devices was determined to be R2 =
0.8351. Figure 1 shows all the data points plotted with a fitted linear line. Note that all data are presented as mean standard
deviation.

Cell Density from Scepter (cells/mL)

Hemocytometer v. Scepter
1.20E+06
1.00E+06

y = 0.0622x + 40015
R = 0.8351

8.00E+05
6.00E+05
4.00E+05
2.00E+05
0.00E+00
0.00E+00

5.00E+06
1.00E+07
1.50E+07
Cell Density from Hemocytometer (cells/mL)

2.00E+07

Figure 1 Class data for the hemocytometer and Scepter were pooled together and plotted. The fitted line equation and correlation
coefficient R2 are given in the plot above. Red markers indicate Scepter values obtained using a 40 m sensor while blue markers
indicate the use of a 60 m sensor.
Interpretation of Results
While the results of this trial were not very reassuring, the pooled results show the strength of the hemocytometer in measuring
accurate cell density. In this experiment, the live cell density measures differ by a 43.2% difference and thetotal cell counts differ by
37.8% between the two methods. The most likely explanation for these differences was a lack of uniform suspension of the cells in the
original sample. Three measurements were necessary with the hemocytometer because the cell counts differed by more than 10%, so it
is reasonable to assume the cells were not evenly distributed in this suspension or in the following suspension from which the
hemocytometer sample was extracted. The lack of a uniform suspension could definitely create a situation where more cells are
present for hemocytometer cell counting compared to Scepter cell counting leading to high cell density variance for the two methods.
Two other conceivable causes of the difference in these numbers are the possible presence of bubbles in the Scepter and the possibility
of unlabeled dead cells in the hemocytometer counts. Both of these would have resulted in a lower live cell density for the Scepter
than the hemocytometer, as observed.
The pooled results from 17 trials, however, resulted significantly better with a nearly one to one correlation and an R2-value of 0.8351
from this correlation. A perfect one to one correlation has an R2-value of 1, but accounting for a range of errors in the technique of
different groups and the inherent errors of the experiment as described above, a value of 0.8351 is reasonably promising and signifies
that both methods produce comparable counts. It is worth noting that although only three trials made use of the 40um sensor in the
Scepter, this sensor resulted in more accurate and precise results.
The size distribution of our 3T3 cells compared with that of Millipores 3T3-L1 cells was good (Millipore.com). This experiments
data peaked between 12 and 19um cell diameter, and Millipores peaked between 13 and 18. To the left of our main peak there was a
smaller peak; this is observed in Millipore data as well. These similarities are a good sign that our cells were the correct size.
These two methods effectively counted the cell density of a given sample. One drawback of the Scepter is that it lacks the ability to
give a percentage of cell viability for a sample. Drawbacks of a hemocytometer include the time taken and the much larger
contribution of human error to the results. When compared with a Coulter Counter, a Scepter is a much more reasonably sized and
convenient. More generally, cell counting is used for transfection experiments, the preparation of qPCR experiments and for splitting
cell cultures to maintain sufficient media. Beyond this, accurate and precise cell counting is particularly important to TEMP
companies, so they dont distribute an incorrect amount of product; the scepter would save them a great deal of time. As the field of
tissue engineering advances, the need for a fast, convenient instrument to count cells will become increasingly important; thus,
compared to the lengthy and varying counts of the hemacytometer, the Scepter is bound to become a tool that is indeed indispensable.