FACSAria SORP Standard Operation Protocol Basic Operation SORP Standard... · 2016-01-08 ·...

Faculty Core Facility, Li Ka Shing Faculty of Medicine

BD FACSAria SORP Operation Manual

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FACSAria SORP Standard Operation Protocol

Basic Operation

1. Checking Lasers Status

a. Click on BD Coherent Connection program to ensure the laser(s) are

“ON”. Lasers warm up duration ~ 30 minutes.

b. Turn off the lasers that are not required for analysis and/or sorting.

If only channels of FITC, PE, and APC are required; the UV and

Violet lasers can be switched off.



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Power of Blue laser is NOT optional. It is ON, together with the

equipment.

Switch off UV laser if it is not required. UV laser excites at

~355nm, it may cause DNA damage during sorting experiment.

Make sure the laser output of UV laser is set at 5.0 before laser is

turned “ON” or “OFF”. You may refer to the following steps.

To switch OFF UV laser,

Output must first be reduced to 5.0 mW before turning off

the laser



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2. BD FACSDiva Software Log In

a. Log into FACSDiva software with your own login name and

password. Please contact the administrator to establish a new user

account.

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

located.



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3. For Sorting: Stream Break-Off and Droplet Formation (100µm

nozzle)

a. Turn ON the Stream and allow time for it to

stabilize.

b. Adjust the Frequency (Freq) between 27 and 30

to identify the HIGHEST break-off, where the

End-portion of the breaking stream can still be

observed.

c. Adjust the Amplitude (Ampl) to introduce DROP 1

measured between 120 and 180; but repeat the

search using a different Frequency if the finishing

Amplitude value is greater than 20.

d. Fine tune the Amplitude value to stabilize the

GAP measured between 8 and 12.

e. Ensure that the satellite droplet has completely

merged with the trailing drop (before

disappearing).

f. Match the input number with the measured value

in DROP 1.

g. Allow the stream to stabilize for a minimum of 3

minutes before the activation of Sweet Spot.

h. Ensure the stream has been stable for at least 3

minutes and that the Sweet Spot is turned ‘ON’.



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Drops Too FEW Drops Too MANY Break-off Too SOON

Solution: Increase Freq Solution: Decrease Freq Solution: Decrease Ampl

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Gap Too NARROW Gap Too WIDE Break-off Too LATE

Solution: Increase Ampl Solution: Decrease Ampl Solution: Increase Ampl



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4. For sorting: ACCUDROP Drop Delay Test (100µm nozzle)

a. Turn on Voltage Test Sort move voltage sliders to appropriate

positions appeared similar to below.

DO NOT touch the deflection plates when the plate voltage is

ON!!! (When the voltage warning light illuminates, indicating that the

plates are charged at 12,000-volt).

b. Adjust the Micrometer Dial to obtain evenly bright bead spots for the

four side streams.

Core Stream Bead Spots TOO BRIGHT Bead Spots UNEVENLY BRIGHT

c. Use the existing well-prepared Accudrop bead solution in the fridge.

You are allowed to prepare fresh stock only if Accudrop solution has

been used up (1 drop Accudrop beads + 350µL FACSFlow solution).

d. Go to Browser Window Experiment New Experiment

Accudrop Drop Delay OK.



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e. Click “+” of Specimen_001, and set the tube pointer onto Tube_001.

f. Make sure the flow rate is 1.00 followed by loading sample into the

Bulk Injection Chamber.

g. Adjust the flow rate to achieve ~1200 events per second; if failed,

increase the flow rate or prepare suspension at a higher concentration.

h. Go to Browser Window ‘Create a new Sort Layout’.

i. Select ‘2 Tube’ in Device ‘Initial’ in Precision right click on

the Left Position and select ‘Add > P1’.



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j. Click Voltage and Optical Filter on the Side Stream Window Sort

on the Sort Layout Window.

k. Click Cancel on the Confirm Prompt.

l. Adjust the voltage slider and make sure the left side stream is

contained in the box.

m. Adjust the Drop Delay value (either or ) to achieve ~100%

intensity in the left side stream.



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n. When proximate to 100% intensity, change to ‘Fine Tune’.

o. Optimize the drop delay again (either or ) until the left side stream

intensity is consistently > 95%.

Note: Two percentage values must add up to 100 %.

p. Turn off the Voltage and Optical Filter.

q. Click Sort to stop sorting; select Cancel when asked if preferred to

save the report. Unload the tube.



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5. Sample Collecting Tube(s) Alignment (FOR SORTING)

a. For sorting, lift up the lid and insert the mock tubes into appropriate

collection device. Holders are available for 1.5mL microtubes,

12x75mm tubes, and 15mL Falcon tubes.

b. To keep the collecting tubes cool throughout the sorting process,

collection device should be connected with tubings directed from the

water bath.

c. Click Voltage Test Sort Waste Drawer Open the sort

block door Adjust the slider of the required side stream and

locate the position for target cells collection.

d. Turn off Voltage Test Sort Waste Drawer Close sort block

door when alignment has been completed remove the mock tubes

and replace new collection tubes with fresh media.



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6. Creating A New Experiment

a. Browser Window ‘New Folder’ ‘New Experiment’.

b. Select ‘Cytometer Settings’ ‘Parameters’ ‘Delete’ the

unnecessary parameters on the Inspector Window.

c. FSC (measures Cell Size) and SSC (measures Cell Granularity) are

MUST for all kind of analysis; their ‘Area’, ‘Height’ and ‘Width’ but

‘Log’ boxes should not be ticked. ‘Log’ and ‘Area’ boxes of

fluorescence channels must be ticked except for cell cycle and/or

DNA analysis.



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d. 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.

e. Select ‘New Specimen’ expands the Specimen to show Tube 001.

Highlight the tube with the Tube Pointer.

f. Select ‘Dot Plot or Histogram’ move the cursor onto the blank

worksheet.



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g. Right click on a plot ‘Duplicate’ to create another plot of the same

type with identical axis.

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



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i. Below shows a template of plots used in routine analysis.

Dot Plot Dot Plot Dot Plot

Dot Plot Dot Plot Dot Plot

Histogram Histogram Histogram



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j. Right click on the plot Show Population Hierarchy

Right click on the plot Create Statistics View right click on the

statistics view Edit Statistics View

k. Select Statistics tab tick mean of FSC-A and FSC-H tick the

mean of the ‘A’rea of the parameters click OK



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7. Procedures for sample acquisition

a. Put your sample tube onto the loading dock. Ensure that the flow rate

is 1.0. Press ‘Load’ on Acquisition Dashboard.

b. Identify the population of interest by adjusting the voltage of FSC and

SSC on Parameters Press Restart to accelerate the changes



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c. Cytometer LaserAdjust the FSC Area Scaling 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.

d. Adjust voltage of each of the fluorescent channels; preferably the

peak of population of interest is greater than ZERO but less than 102.

e. Press ‘Unload’ on Acquisition Dashboard to unload the current

sample tube.

f. 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.



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8. Creating Gates

a. Set the current tube pointer to the following tube and repeat step l, m,

o and p with the Unstained / Negative Control tube.

b. Gate the cells of interest according to the following sequence by using:

Autopolygon

Polygon Quadrant Interval Snap-to

Rectangle

Highlight P3 population under P2

Create a polygon gate (P1) to

gate the main population on

the FSC-A vs SSC-A dot plot

Highlight P1 population in “Population Hierarchy’

Create a polygon gate (P2)

around the singlet event on the

FSC-H vs FSC-W dot plot

Highlight P2 population under P1

Create a polygon gate (P3)

around the singlet event on

the SSC-H vs SSC-W dot plot



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c. To define fluorescence positive signal, interval gate (P4, P5,…) beyond

negative peak of fluorescent channels in histogram plot of unstained

samples can be created. For over multiple fluorescence channels,

quadrant gate could be created to define single/double positive signals

(Q1; Q2; Q3; Q4….).

*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, & P6 populations. On the hierarchy table,

you 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, P5, & P6 populations are drawn.



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d. Acquisition Dashboard click Next Tube to create a slot for new

sample tube / to assign to the following sample tube slot.

e. Ensure the Acquisition Setup has been arranged as follows:

f. Acquisition Dashboard click Record Data to record entire sample set.



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9. Setting up a Sorting Experiment

a. Ensure that the flow rate is 1.0 before loading the sample tube.

b. Browser Window ‘Create a new Sort Layout’.

c. Determine the most appropriate options from Device and Precision

on the Sort Layout Window.

(i) For 2 - 12x75mm tubes sorting, select ‘2 Tube’ device

(ii) For 4 – 15mL Falcon tubes sorting, select ‘4 Tube’ device

(iii) To achieve the highest yield sorting, choose ‘Yield’

(iv) To achieve the highest purity sorting, choose ‘Purity’ in ‘2

Tube’ sorting and ‘4-way Purity’ in ‘4 Tube’ sorting.

(v) To achieve single cell sorting, choose ‘Single Cell’



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d. Right click Far Left / Left / Right / Far Right Position ‘Add’

Gate of Interest Sort

e. Click OK on the Confirm Prompt.

f. Acquire data for the entire sample set. In each run, record all data by

clicking ‘Record Data’.

g. Constantly check for the sufficient level of samples inside the loading

chamber and plenty of room inside the collecting tubes for sorted

samples. Unload the sample tube and rinse the tubing with

FACSRinse® solution and Milli-Q water for five minutes each, if the

threshold rate fluctuates high and low, and/or the stream is unstable.



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Notes – During the process of Sorting

1. To help achieve an optimal result, users are advised to pay attention to

the following items:

Sample: 5 tubes of 1mL sample, rather than 1 tube of 5mL sample

Threshold Rate: Stable and no fluctuation

Sort Rate: Stable and no fluctuation

Stream: Stable and no sudden movement

Side Stream: Stable and no fanning

Sample Tube: Sufficient sample level to prevent clogging

Collecting Tube: Plenty of room for collection

2. If required to change the collecting tubes ‘Pause’ retrieve the

fully filled collection tubes and replace with new tubes ‘OK’ on

the Confirm Prompt ‘Resume’

3. Upon completion, click ‘Sort’ ‘OK’ ‘Unload’ the sample tube.

10. Cleaning the System

a. To 3 x 5mL tubes add 2 ml of FACSClean® Solution, FACSRinse®

Solution, and Milli-Q H2O.

b. Load each tube into the loading chamber.

c. Increase the flow rate to 11.0 and let each solution run for 5 minutes.

d. Repeat the same with FACSRinse® Solution for 10 min, if Propidium

Iodide (PI) is used.



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11. Export FCS Data / Experiment

a. To save Experiment, right click on the ‘Experiment’ ‘Export’

‘Experiment’ ‘Browse’ to choose the destination folder.

b. Create a new folder and rename, then click ‘Export’ ‘OK’.

c. To save FCS files, right click ‘Specimen’ ‘Export’ ‘FCS

Files’.



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d. Select FCS 3.0 ‘OK’ ‘Browse’ to choose folder destination.

e. Create a new folder and rename ‘Choose Directory’ ‘Save’.

f. Delete experiment after having FCS data / experiment exported.

12. Log Out

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

FACSAria SORP Standard Operation Protocol Basic Operation SORP Standard... · 2016-01-08 ·...

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Transcript of FACSAria SORP Standard Operation Protocol Basic Operation SORP Standard... · 2016-01-08 ·...