6 weeks at SIP (Time sure flies)

Hey guys! Time sure pass with the speed of light, it’s already the end of 6th week! And it’s my turn to present to everyone this week.

Hope everyone is still doing well with your SIP and MP! =)

Today, I am going to be presenting on what I usually do in my SIP:
1. Daily Quality Control (QC) for FACSAria
a. This has to be done everyday to verify that the FACSAria is in optimal working condition

This involve 3 main steps:
1. “On” the computer before the FACSAria
a. 1 cycle of “fluidic startup” (primes the system of bubbles)
2. “Time delay” QC calibration (optimization)
3. “Area scaling” QC adjustment

Since step 1 is self-explanatory, I will skip straight to step 2:

Time Delay

· “Time delay” is required to adjust so that the signals obtained from the firing of different lasers at different timing are recognized to be from the same cells by the computer.
(Refer to figure 1, below)
· “Time delay” QC calibration is required to be done for all lasers, except for blue laser, which acts as a reference.
(In a flow cytometer, it is usually standard to have the red and blue lasers)



Steps:
1. Load a tube of DI water into the FACSAria to clean the sample line (Flow rate: 10)
2. Prepare a tube of approximately 500uL of rainbow beads solution
a. Rainbow beads contain mixed fluorescent signals (eg. FITC, PE and etc)
3. Load the rainbow beads tube into the FACSAria (Flow rate: 1)
4. Gate the cluster of dots on the dot plot for singlets
a. Singlets are always located on the low SSC and low FSC on the graph
(Shown below in figure 2)



5. Once “gated”, change the window extension to 0
6. Start optimizing the “time delay” of the laser by getting the peak of the signal as right as possible
a. If the value of “time delay” is not optimized, the peak of the signal is always to the left. (refer to figure 5)
b. After the “time delay” is optimized, change the window extension back to its original value



Window extension:
This relates to the pulse measured, as the pulse measured from the
beads is quite narrow, window extension is set to 0 so to capture
only the data of the pulse signal from the beads.





Area Scaling

· Area and height mean value must approximately match for all parameters (eg. FSC, SSC, FITC, PE and etc)
o Producing a linear line on the FSC-Area vs FSC-Height dot plot
· This is to standardize the parameters for accurate interpretation of experiment results.
· “Area scaling” is required to be done for all lasers.



Steps:
1. Using the same tube of rainbow beads, load it into FACSAria (flow rate: 1)
2. “Gate” the cluster of dots which represents the singlets
(Shown below, figure 8)



3. Adjust the values of the area scaling to increase or decrease the mean area-value to match approximately the mean height-value of the signals acquired using the population statistics (FSC-A, SSC-A, FITC-A and etc)
(Refer to figures 9, 10 and 11)
4. After the mean area to height value has been approximately matched, record the data of 1000 events by clicking the “Record Data” button on the “Acquisition Dashboard”.
(Refer to figure 12)









=== Sorry for the late posting, need to make sure I get my supervisor permission first before posting ===

Posted by:
Low Quan Jun
0607243C
TG02
Group 08
05 August 2008