OBJECTIVES: 1. To know the maintenance technique of establish cells. 2. To be able to do the cells counting by using a Hemocytometer. 3.

To count the cells. 4. To evaluate the viability of the cells.

INTRODUCTION:

Cell maintenance workstations automate the major bottlenecks of cell-based assays, such as media exchange, cell plating, incubation and assay preparation. HL-60 cells require simple maintenance in vitro and grow as single-cell suspension cultures without the tendency to adhere to the flask. HL-60 cells require simple maintenance in vitro and grow as single-cell suspension. Doubling times are around 24 h in an actively growing culture. Cultures can be maintained by diluting the cells with a fresh medium. While the HL-60 cell line is very simple to culture, it is predisposed to differentiate into nonproliferating cells or to a subline. Thus, meticulous attention to its handling, culture, and passage procedures is required. Cell concentration should not be allowed to exceed 106 cells/ml. Cell counting and evaluation of the viable cells is done to measure performance, to achieve reproducibility, or to make comparative studies, a means of quantifying the cell population is need. The growth of mammalian cells in culture can be monitored by a number of parameters related to the increase of cellular biomass over time. The simplest method is by cell counting at regular intervals. The system of viability assay for mammalian cell culture can be applied to the determination of cell viability for engineered tissue. The most common routine method for cell counting which is efficient and accurate is with the use of a hemocytometer. Hemocytometer is a device originally designed for the counting of blood cells. It is now also used to count other types of cells as well as other microscopic particles. To measure the viability of the cell, Trypan blue is used. The trypan blue dye exclusion assay is the most commonly used and accepted method for the measurement of cell viability. It relies on the alteration in membrane integrity as determined by the uptake of dye by dead cells, thereby giving a direct measure of cell viability.

RESULTS:

Corners of gridlines

Figure 1 : Hemacytometer gridlines

CELL COUNTING FORMULA 1) C = Av x 2* x 104 cells/ml C = cell concentration (cell/ml) Av = average number of cells in four corners counted *2 = dilution factor

Av = 101 + 112 + 111 + 118 4 = 442 4 = 110.5 C = 110.5 x 2 x 104 = 2.21 x 106 cell/ml

2) Percentage of viability (%) = Nv / NT x 100 Nv = number of viable cells NT = number of total cell population (%) = 442 x 100 442 = 100 %

DISCUSSION:

In this experiment, we have to do the cell counting and evaluation of the viable cells. However, to do that, we have to check for the maintenance of the cells. First of all, the cultures were examined whether there was a contamination or not. If there was any contamination, the maintenance of established cell cannot be done. In suspension culture, no need to use trypsinization because the cell did not attach to the flask. The cell was resuspended carefully to homogenize cell suspension. The cell then centrifuges using 2000rpm for 5 minute. All the supernatant were discard out and let the pellet in the flask. Then, fresh medium was added. When using the lower volume of the media, the concentration of cell will high. In the flask, the cell was dispersing by repeated pipeting.

Before the cell counting was done, the slide was prepared first by cleaning the slide with 70% alcohol. This was done carefully to avoid the scratching of the semi silvered surface of the slide. Then a clean coverslip was pressed down to attach over the grooves of the counting area. On the other part, the cell suspension was resuspended thoroughly. 20L of the suspension cell was collected and then was placed onto a clean surface followed by the addition of trypan blue dye. Then both of the solution was well mixed to avoid formation of bubbles. Trypan blue dye was applied for the measurement of cell viability. It relies on the alteration in membrane integrity as determined by the uptake of dye by dead cells, thereby giving a direct measure of cell viability. After that, the cell suspension was transferred immediately to the edge of the hemocytometer chamber and then it was viewed under microscope for the cell counting. The viable cells were identified as shiny and in intact form whereas the non-viable cells were identified as blue colonies due to the uptake of the trypan blue dye. The cells were counted within the four corners of the gridlines as shown in figure 1(result). From the results, the cell concentration was 2.21 x 106 cell/ml. The percentage of the viability was 100%. This is because all the cells were still viable during the cell counting and this gave us 100% cell viability.

CONCLUSION:

From this experiment, we were able to know the technique for maintenance of established cell which involved identification of the contamination, the cells confluence and the cells morphology. Plus, we also be able to perform the cells counting by using a hemocytometer as well as the evaluation of the viable cells. From the result, the cell concentration was 2.21 x 106 cell/ml and the percentage of the viable cells was 100%.

REFERENCES:

1. http://www.tecan.com/platform/apps/product/index.asp?MenuID=3009&ID=5900&Men u=1&Item=33.29.8 2. http://toxsci.oxfordjournals.org/content/76/2/376.full#ref-6 3. http://www.abcam.com/index.html?pageconfig=resource&rid=11454 4. http://tools.invitrogen.com/content/sfs/appendix/Cell_Culture/Viable%20Cell%20Counts %20Using%20Trypan%20Blue.pdf