Faculty Core Facility Version 1.1 LKS Faculty of Medicine ... protocol34.pdf · Faculty Core Facility Version 1.1 LKS Faculty of Medicine BD FACS LSRFortessa Operation Manual 3 Select

Faculty Core Facility Version 1.1

LKS Faculty of Medicine

BD FACS LSRFortessa Operation Manual

1

FACS LSRFortessa Standard Operation Protocol

Basic Operation

Check sheath tank and waste tank

Please empty waste tank if liquid reaches to the red line (Please refer to attachment 1 on page

16: SOP for empty waste tank. ).

Please refill sheath tank if its weight is less than 7KG (Please refer to attachment 2 on page

17: SOP for refill sheath tank).

Launching the BD FACSDiva Software

Log into FACSDiva software with your own login name and password. Please contact the

administrator to create a new user account.

When connected, click ‘Use CST Settings’ when a mismatch was located.




2

Creating and working with experiment in BD FACSDiva Software

On the Browser toolbar, click the New Folder button to create a folder. Rename the folder if

necessary.

On the Browser toolbar, click the New Experiment button to create an experiment. Rename

the experiment if necessary.

Select Cytometer Settings and remove the unnecessary parameters on the Inspector Window.




3

Select appropriate parameters by checking their corresponding boxes. FSC (correspond to the

Cell Size) and SSC (correspond to the Cell Granularity) are MUST for all kind of analysis.

Both FSC and SSC ought to be displayed in linear scale, hence their ‘Log’ boxes should not

be ticked. “Log” must be checked for fluorescence channels except for cell cycle and/or DNA

analysis. Users are strongly advised to check the boxes in respect to the ‘H’eight and the

‘W’idth of FSC and SSC.

Choose Experiment > Experiment Layout, and define labels for each parameter. Select the

column of fluorescence channel and enter a label in the Quick Entry Label field.




4

Creating plots on the global worksheet

On the Browser toolbar, click ‘New Specimen’; then expand the Specimen to show Tube 001.

Highlight the tube with the Tube Pointer. Rename the specimen if necessary.

From the option bar select ‘Dot Plot / Histogram’; then move the cursor onto the blank

worksheet and drag the plot to an ideal size.




5

Right click on a plot and select ‘Duplicate’ to create another plot of the same type with

identical axis.

Select each individual axis, and opt from a list the preferred parameter.

Below shows a template of plots used in routine analysis.




6

Right click on the plot and select ‘Create Statistics View’; then right click on the statistics

view and select ‘Edit Statistics View’

Select the third tab, Statistics, and check the mean of FSC-A and FSC-H

Also check the mean of the area of the parameters of your choices

Select the first tab, Plot, on the Inspector Window; and check the corresponding box if you

wish an exponential display for the axis on particular plot(s).




7

Press “RUN” and “LO” on fluid control panel.

Gently tab the tube to mix your sample, then put your sample tube on SIP (sample injection

position); Run the unstained sample before other sample tubes.

On Acquisition Dashboard, click Acquire Data .

Identify the population of interest by adjusting voltage of FSC and SSC on Parameters tab of

Cytometer.

(Press ‘Restart’ to accelerate the changes to be shown on the dot plots and

histograms)

Adjust the FSC Area Scaling on Laser tab of Cytometer, until the mean of FSC-A and FSC-

H are APPROXIMATELY SAME. Return to Parameters tab and finely re-adjust voltage of

FSC and SSC. Your population of interest should be displayed in the center of FSC v SSC

dot plot.

Adjust voltage of each of the channels; hence their signal peaks sit next to the Y-axis of

the histograms, with majority of the peak from the population of interest greater than

ZERO.




8

Click Stop Acquiring on Acquisition Dashboard and replace your sample with DI H2O.

Repeat above steps with the positive control sample tubes. Adjust the voltage of

corresponding channels if their signal peaks are outside the limit of the histograms.

Creating Gates

Gating tools:

Set the current tube pointer to the Unstained Control tube.

Right click on the plot and select ‘Show Population Hierarchy’.

Create a polygon gate (P1) around the singlet events in the FSC-A vs SSC-A dot plot.

Highlight P1population in “Population Hierarchy’.

Autopolygon

Polygon Quadrant Interval Snap-to

Rectangle




9

Create a polygon gate (P2) around the singlet events in the FSC-H vs FSC-W dot plot.

Highlight P2 population in “Population Hierarchy’.

Create a polygon gate (P3) around the singlet events in the SSC-H vs SSC-W dot plot.

Highlight P3 population in “Population Hierarchy’.




10

To define fluorescence positive signal by creating interval gate (P4, P5,…) beyond

negative peak of fluorescent channels in histogram plot of unstained samples. For over

multiple fluorescence channels, quadrant gate could be created to define single /double

positive signals (Q1; Q2; Q3; Q4….)

Note that P1 is the children of All Events and the parent of P2 population; P2 population is the

children of P1 population and the parent of P3 population and the grandparent of P4, P5

populations. Thus, on the hierarchy table, users should highlight the P1 population when a gate

for P2 population is drawn, the P2 population when a gate for P3 population is drawn, and the

P3 population when gates for P4 and P5 populations are drawn.

Click the New Tube button to add more tubes. Rename the tubes if necessary.




11

Recording data for all samples

Ensure the Acquisition Setup has been arranged as follows:

Press “RUN” and “LO” on fluid control panel.

Gently tab the tube to mix your sample, then put your sample tube on SIP (sample

injection position); Run the unstained sample before other sample tubes.

On Acquisition Dashboard, click Acquire data and

Record data

Please pay attention to the sample tube to make sure it will not run dry!

Click “Stop Acquiring” to stop acquire the data if necessary then unload your sample.

Repeat above steps for each sample.




12

Cleaning the System

To do cleaning step by step (HI, 3, 1, 5, 2, DI):

Cleaning procedure between each user is required.

• Press RUN and HI on the cytometer fluid control panel.

• Prepare 3 mL of cleaning solution (FACS Clean, FACS Rinse, DI H2O)

• Install a tube of FACS Clean solution on the SIP with the support arm to the side

(vacuum on) and let it run for 1 minute.

• Move the tube support arm under the tube (vacuum off) and allow the cleaning solution

to run for 5 minutes with the sample flow rate set to HI.

• Repeat steps 2 and 3 with BD™ FACSRinse solution.

• Repeat steps 2 and 3 with DI water.

• Remove the DI water tube from the SIP.

• Move the tube support arm to the side.

• Press the PRIME button on the fluidics control panel.

• When the STNDBY button lights (amber), press the PRIME button again.

• Place a tube containing 1 mL of DI water on the SIP.

• Press the STNDBY button on the fluidics control panel.




13

Export Data

Right click on Specimen in Browser Window; move down to select Export > FCS Files.

Select FCS 3.0 in File Version; and click ‘OK’.

Click ‘Browse’ to choose the destination folder as: D\User\Your department\Your

name\Date.




14

If necessary, create a new folder and rename accordingly.

Click ‘Choose Directory’ when finish.

Click ‘Save’ to export FCS files.

Click “Export” >“Experiment” in “File”

Click ‘Browse’ to choose the destination folder as: D\User\Your department\Your

name\Date.

Click ‘OK’ to export Experiment.

Delete Your Experiment after data export.




15

Log Out

To log out of FACSDiva software, go to File > Log Out.




16

Attachment 1

BD FACS LSR Fortessa Standard Operation Protocol

--- Empty Waste Tank




17

Attachment 2

BD FACS LSR Fortessa Standard Operation Protocol

--- Refill the sheath container

Documents

Faculty Core Facility Version 1.1 LKS Faculty of Medicine ... protocol34.pdf · Faculty Core Facility Version 1.1 LKS Faculty of Medicine BD FACS LSRFortessa Operation Manual 3 Select