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.
Gwen,
ReplyDeleteCell concentration quantitation using the hemocytometer:
- Which squares do you count?
- Did you use trypan blue to differentiate between living/dead cells? What is your dilution factor?
- You stated "Media (twice the amount of trypsin added)" in your post. Therefore how many times dilution is it in the end for trypsin.
- I noticed that you stated that cell counts are averaged. So how many times did you count the cells?
- Is there other methods/technique that I can count the cells more quickly than using the hemocytometer?
Li Yinliang Alex
TG02 0704894E
Group 8
16 July 09
Hi Alex,
ReplyDelete1)Like what we did in school, i too counted the five squares on the hemacytometer.
2)No i did not use tryphan blue as there was no need to differentiate between the dead and living cells. The main reason that the cells are counted is to determine the amount of media to be added so that the desired seeding concentration is achieved. In addition, i mentioned that i washed the flask with PBS. This not only removes the serum in the media that may inactivate trypsin, it also washes away any dead cells.
3)Pertaining to my statement, what i meant was that if you add 1ml of trypsin, 2ml of media must be added to neutralise its effects. So usually 1-3ml of trypsin will be added and 2-6 ml of media used to stop the trypsinisation effets. It is not really a dilution factor thing.
4)As i mentioned earlier, 5 squares were counted. If you remember from MCT and hematology, we too count the cells from all 5 squares and then do an average by dividing the total cell number by 5.
5)I am sure there is a faster way to count the cells but for my case, it need not be a very detailed count, just to know how many cells there are to achieve the desire seeding concentration.
I hope I have helped clear your doubts. If you still have certain doubts, feel free to approach me.
Thanks,
Gwen
Hello Gwen,
ReplyDeleteThe title of your post is "Flow Cytometry"....but there is nothing about it in your posting.