Flow Cytometry

Hi everyone, its Gwen here and this week, it’s my turn to share about my SIP experiences. I am in a very pleasant work place and the people here are very friendly. I have also learnt many new laboratory techniques and processes. My research laboratory is doing research of using nano particles as delivery vectors to treat cancer. It aims to reduce the toxicity effects of the drug and to increase the delivery of the therapeutic agents to the tumour. My job basically involves work in the cell culture room. Although I have learnt cell culture techniques before, but during my attachment, I have learnt many new techniques and improved my pipetting skills. In today’s entry, I will be talking about flow cytometry and its preparation steps.

Flow cytometry can be done to test for various purposes. In my case, it is to investigate the cellular uptake efficiency of the various treatments that will be mentioned later.

Firstly, the cells must be in confluence so that they can be seeded on to 12-well plates. For seeding, the cells in the flask first need to be trypsinised. So, the used media must first be removed and the flask must be washed with PBS. This is to remove any residual media that may inhibit the effect of trypsin. Next, trypsin must be added and then the flask is incubated for 5 mins at 37°c. After 5 minutes, the flask is viewed under the microscope to ensure that the cells are no longer in clumps and are single-celled. Media (twice the amount of trypsin added) is then added to the flask and pipetted up and down to inhibit the action of trypsin. If the cells (previously seen under the microscope) are still in clumps, the media would be pipetted more vigourously against the wall of the flask. After ensuring that the clumps are gone, the cells are then pipetted into a 50 ml falcon tube.

10µl of the cells are then pipetted into a hemocytometer and the number of cells is counted, and then averaged. This is to determine the amount of cells and fresh media that is to be added to each well to achieve the wanted cell concentration. After the total amount of cells and fresh media have been calculated for all the wells to be seeded, the calculated amount of cells and fresh media are added into a new 50 ml falcon tube and pipetted up and down to mix it well. The cells (with fresh media) are then pipetted/poured into a reservoir. I ml of the cells are then pipetted into each well of the 12-well plate. The plates are then incubated overnight. Throughout the whole process, it is very important that there must be no bubbles present as the bubbles can cause asphyxiation. The remaining cells left are subcultured into a new flask to continue the cell line.

The next day, treatment is prepared according to a table that my mentor provides. The table determines the amount of polymer, apoptotic protein, drug, buffer and media to be added based on the different conditions and/or concentrations that were set by her. For example, the condition can be addition of the free drug, the drug with the polymer, the apoptotic protein only, the apoptotic protein with the free drug and the apoptotic protein with the polymer and drug. For flow cytometry, the drug and apoptotic protein contain fluorescent dyes that are highly sensitive to light, thus all the lights in the cell culture room must be turned off. The laboratory that I am working at deals with certain light sensitive drugs and I had an experience of working in total darkness except for a torch light. It was really an unforgettable incident. After adding the treatment to the cells, the plates were incubated for another 3 hours at 37ºc.

After incubation, the cells will then be washed with PBS and then detached with trypsin. The effect of trypsin is then neutralized with PBS and the cells are pipetted to a 15 ml falcon tube. The cells are then centrifudged at 1500 rpm for 5 minutes, with the temperature set at 4ºc to prevent degradation of the cells. The supernatant discarded and the cells are resuspended with PBS and then transferred to two polystyrene flow cytometry tubes. The tubes are then stored in an ice box and sent for flow cytometry. The flow cytometry machine is found in a shared facilities area and the machine is operated by a trained technician.


Taken from:http://www.eppendorf.com/int/index.phppb=072836f7d878666d&action=products&contentid=1&catalognode=9537
This is the multichannel pipette that i mention above.


Taken from:www.bcm.edu/cbass/?PMID=8386
This is the flow cytometry machine that i saw.


Taken from:www.medsci.org/v06p0051.htm
This is an example of how a flow cytometry result will look like but it is not my result.

That’s all I have to share this week, see you all again next time.