This lab will depict the culturing of adherent HEK 293 cells and the counting of cells through the use of a hemocytometer in tandem with a phase contrast microscope.

Materials

Starting Confluency Check

Prelab Preparation

Please refer to lab 1: Making MEM media for Biosafety hood and water bath setup

Culturing Cells

After prelab preparation, turn on the peristaltic pump and sterilize the vacuum hose with 70% EtOH. Then utilizing the sterile technique grab one pasture pipet from the container as shown above. With the pasture pipet inserted into the vacuum hose remove the excess media from the culture plate.

Using a 5ml pipet draw up 4ml of Trypsin/EDTA and eject it into the culture plate.

With the Trypsin/EDTA in the culture plate, place the plate back in the 37°C incubator for 5 minutes. This allows the Trypsin to remove Ca++ molecules from important cellular junctions and release the adhered cells from the culture plate.

While the culture plate is in the incubator, MEM media can be added to the falcon tube and four plates that will be used to split the cells. 10ml of media will be transferred into every container.

The falcon tube was then transferred to the centrifuge where it was balanced with an equivalent volume across from the tube. The centrifuge was then set at the 30 speed for 3 minutes. After the 3 minutes a cell pellet was visible at the bottom of the tube. Before transferring the tube back into the Biosafety cabinet the setrile technique was observed by rinsing the tube with 70% EtOH.

Using a pasture pipet the excess media was removed until there was around 100microliters of media surrounding the cell. After which 8ml of media was transferred into the falcon tube. The cell pellet was also gently titrated during this process, to resuspend the cells.

Hemocytometer and Counting Cells

Prior to using the hemocytometer, it and the coverslip were cleaned using 70% EtOH over a petri dish. This was done to avoid dropping and breaking it.

Utilizing a pipetman, 20microliters were transferred to wedge located in the center of the hemocytometer, extending under the cover slip. The hemocytometer was then placed under the under the microscope and the four corner grids were located. The cells in these grids were counted using a laboratory counter.

1750microliters of cell suspension was added to both of the remaining fresh culture plates. This yields a 500k cells per plate split At the end of this experiment, the newly split culture plates were then placed into back into the incubator and observed closely for a few days after the experiment noting adherence and growth.