FACSAria SORP Standard Operation Protocol Basic SORP Standard... · PDF file...

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  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    1

    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.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    2

     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

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    3

    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.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    4

    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’.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    5

    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

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    6

    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.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    7

    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’.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

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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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    BD FACSAria SORP Operation Manual

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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.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    10

    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.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    11

    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.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

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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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    BD FACSAria SORP Operation Manual

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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.

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    14

    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

  • Faculty Core Facility, Li Ka Shing Faculty of Medicine

    BD FACSAria SORP Operation Manual

    15

    j. Right click on the plot  Show Population Hierarchy