Week 16: 4th Official Blog Posting

Hi guys & gals!

Woah, time sure flies, I can still remember the first day of my SIP, but it's now left with 4 more weeks of SIP! Then I can take permenant leave from my supervisor! Can't wait. Hahaz!

Hope everyone is doing well too!

This week, I would like to continue from my last blog posting. Today, I would like to explain how the SP profile is recorded to give a more complete picture to the use of flow cytometer in SP profiling.

For every experiment, the following sample tubes are needed:
1) “Unstained” tube
2) “PI only” tube
3) “Single dye only” stained tubes
4) “Combination of dyes” stained tubes (differ according to each different experiment)

Before recording the data, there are procedures to be done first.

For my experiment, I have the following sample tubes:
1) “Unstained” tube
2) “PI only” tube
3) “Hoechst 33342 only” stained tube
4) “Hoechst 33342 + Verapamil (x) + PI” stained tube
[For simple explanation sake, I will use only one tube with Verapamil as blocker at a fixed concentration (x)]

Firstly, this is what we do:

1) we will take a look at the unstained tube to view the “true negative” and auto-fluorescence stained population

2) Vary the voltages of the “light” detectors to display the desired cell cluster in all the plot graphs

3) View other tubes to take a quick look at our stained population to observe if we have any “positive” stained population

4) Steps 1 to 3 are done interchangeably to bring out our desired cell profile, once done, we proceed to the next step

5) Since I am doing on SP profiling, I will do up a series of gating in the following order:

a. Scatter gate
b. Side Scatter (SSC) gate
c. Forward Scatter (FSC) gate
d. Viability gate
e. Live gate
f. SP gate (To be done after all the cell profiles was recorded)

After we brought out our desired cell profile, we would start recording the data.

Number of events to record (for my experiment only):
1) “Unstained” tube (50K “all event” events)
2) “PI only” tube (50K “viable gate” events)
3) “Hoechst 33342 only” stained tube (200K “live gate” events)
4) “Hoechst 33342 + Verapamil (x) + PI” stained tube (200K “live gate” events)

Note: The number of events to record differs with different operators, different experiments, and ability of the flow cytometer to obtained the desired cell profile

After recording the results, the flow cytometer is prepared to shut down for the day and that’s it.

The operation of flow cytometer differs from operator to operator, however, the recording of tubes always follow a sequence:
1) “Unstained” tube
2) “PI only” tube
3) “Single dye only” stained tubes
4) “Combination of dyes” stained tubes

Please do ask question, about anything you do not understand, I would "love" to explain it to you. Haha.

.....4 more weeks to the end.....

Hope everyone take care of yourself!

Many thanks
Quan Jun
TG02