RII DIVISION FLOW CORE

User Resources & Guides

USER RESOURCES & GUIDES

Below you can click on any of the tabs for cytometer specific user guides, filter layouts, troubleshooting, compensation tips, data management, and downloadable/printable documents.

MGB CORE MANAGEMENT SYSTEM

PREREQUISITE: All users must complete our Orientation process and an Attestation form, prior to gaining access to our core. More information.

    1. Navigate to Research Cores.
    2. New Users –
      1. Click “Sign Up”
      2. Input Partners/MGB ID – Click “Lookup Information” on top right
      3. If you’d like invoices to go directly to a Financial Contact, add “Alternative Email” and change “Send Communication To” to “Both”. (Note: PIs associated with the fund you use will automatically receive a copy of the invoice, regardless of whether you add Alt Email or not)
      4. Verify all information is correct
      5. Click “Create Account”
    3. Existing Users
      1. Log in to MGB CMS account using Partners/MGB credentials
      2. If you’d like to add a Financial Contact to automatically receive invoices, Click “Account” on top right
      3. Add “Alternative Email” and change “Send Communication To” to “Both”. (Note: PIs associated with the fund you use will automatically receive a copy of the invoice, regardless of whether you add Alt Email or not)
      4. Click “Update Account”
    4. Adding Funds to your account
      1. After logging into MGB CMS account, click “User” Menu > “Funds”
      2. Click “+ New Fund” on the right
      3. Enter 6-digit Partners/Peoplesoft Fund Number and click “Lookup”
      4. Verify all information is correct, then click “Assign Fund”
    5. Navigate to RII Division Flow Core Page
      1. Click “Core Services” Menu
      2. Click “Flow Cytometry” link on the left
      3. Click “RII Division Flow Core”
    6. Submit Training Request to access calendar system
      1. Click “Equipment & Pricing” Link
      2. Click “Training Request” button next to the equipment (3L vs 5L) NOTE: you will need to create separate training requests for each instrument
      3. Fill out relevant information
      4. Click “Create this Training Request”
      5. Await response from core personnel to approve Training Request to give you access to the calendar system
    7. Booking an event on the calendar
      1. Click on the link for either “3L Schedule” or “5L Schedule” to view each instrument calendar and availability
      2. Click on the calendar on the specific date and time you wish to book
      3. Adjust times, select specific services, and add a 6-digit Peoplesoft fund or existing fund from the menu to be billed for the experiment
      4. Click “Create”
    8. Modifying or cancelling an event
      1. Click on the experiment block on the calendar that you wish to modify/cancel
      2. Modify: adjust any information and click “Save”
      3. Cancel: click “Cancel Event”
      4. Note: See Cancellation Policy in Rules & Regulations below
    9. Invoices
      1. Invoices will be sent monthly, 7 days after the end of the month when event took place. (e.g. Usage Month: April; Invoices Sent: May 7th)
      2. Review invoices thoroughly and notify core personnel of any discrepancies or changes needed. Core personnel will make changes and issue a new invoice
      3. On the 14th day following the previous months’ service, core personnel will no longer be able to manually adjust previous events, and all changes must be made via MGB CMS administrators (e.g. Usage Month: April, Invoice Changes by: May 14th)
      4. Invoices will be fulfilled electronically and automatically between ~30-60 days after event takes place (e.g. Usage Month: April, Electronic Transfer: May 31st)
    10. RULES & REGULATIONS
      1. Each user must complete our full Orientation & Training process to gain access to the core
      2. Each user is required to have an individual log-in and cannot use a general lab account
      3. Users are allowed to book up to a maximum of 2 hours at a time during “peak hours” (Mon-Fri 12-5pm)
      4. Each user is responsible for making sure the instrument is shutdown or passing that responsibility on to the next user on the calendar. DO NOT assume the next user will show up for their time without having contacted them directly to confirm. Otherwise, when in doubt, shut the instrument down after your usage.
      5. CANCELLATION POLICY:
        1. Users must cancel unused bookings >6 hours before the start time of the event to avoid charges
        2. If a user cancels their booking <6 hours until their start time, and the vacated time goes unused, the user will be charged

Fortessa User Guide

COMPUTER SETUP:

  • Turn on computer before turning on the machine
  • Windows 10 Username: BDOperator
  • Password: BDIS#1
  • Open Tera Term application on desktop and leave running in background
  • Log in to BD FACSDiva software

MACHINE SETUP:

  • Turn on the machine if you are the first user of the day. There is no fluidics startup for the Fortessa.
  • Give the lasers 20-30 minutes of warm-up time.
  • Check tank levels for FACS buffer and waste
    • To fill FACS buffer unscrew probe and place in holster. Silence alarm by clicking button on the wet cart. Fill cube if above 50% full or replace with new cube and screw in probe.
    • For full waste, unscrew probe and holster on side of cart. Empty waste in the sink and refill with bleach to first mark on outside of tank so that a full tank would create a 10% bleach solution. NOTE: make sure to not get the waste cap filter wet. If so, it needs to be replaced immediately.
  • YOU MUST HIT RESTART ON WETCART AFTER REMOVING EITHER PROBE
  • Bleed bubble filter (1st) and sheath line just above filter (2nd) into the beaker to remove any air bubbles caught in the lines and filter.
  • Move sample probe arm to the side, remove the tube and PRIME 2x
  • When red PRIME button goes back to STANDBY, load tube of DI Water, center arm, and RUN for 30 seconds prior to running any samples.
  • Be careful as the probes can be easily bent so only move in a vertical motion.

SAMPLE PREP:

  • You MUST filter samples immediately prior to loading on machine using 70uM mesh or less.
  • Add EDTA to prevent clumping (when possible)
  • Use a lower concentration to run at about 15k events/sec on MED speed.
  • There may be a sample surge of ~100uL when centering the arm underneath the sample. Use minimum starting volume of 250uL.
  • Run 10% Bleach solution every 20-30 samples for 5 mins, then dH2O to prevent clogging

RUNNING SAMPLES:

  • Only load sample on to probe when you are ready to start recording immediately, otherwise leave tube of dH2O running.
  • Samples are aspirated when tube is loaded if arm is to the side whether in RUN or STANDBY, so once tube is loaded immediately center arm.
  • Load sample with machine in RUN and center arm.
  • For control samples, adjust voltages and settings while acquiring.
  • For experiment samples, immediately begin recording once tube is loaded to maximize the event numbers.
  • RUN button turns green when tube is properly pressurized.
  • If the Run button is orange instead of green, it indicates there is a pressure issue. Please check either the tube (cracks on tube), liquid around the top, or air in the air bubble filter
  • The black disc under the sample should have a gap between the sample tube and never be touching as this can cause the probe to bend. If this is the case immediately screw the disc down.
  • Use LO MED HI buttons and SAMPLE FINE ADJ dial to control flow rate.
  • Run samples at a maximum event rate of 15-20k per second. LO speed has best sensitivity and cleanest signals.
  • If samples show events but are pressed up against the FSC axis, likely an air bubble in the flow cell. PRIME 2x with no tube loaded and run DI Water tube for 30 seconds to stabilize stream.
  • If a clog occurs, run 10% Bleach solution until clog is broken down and passed, then dH2O for 5 minutes. If persistent clog, seek help from Flow Core Manager.
  • There is no automatic back-flushing of the outer probe, so wipe down probe with EtOH Kimwipe if worried about carry over or alternatively run a fresh tube of water between samples. (This is only for very rare populations, likely not necessary as carry over is very minimal)

SHUTTING DOWN:

  • After the last sample, run 10% Bleach for 5 mins and dH2O for 5 mins
  • When you are done, leave DI Water tube loaded on the probe and place machine in STANDBY
  • Power down machine or leave on for next user.
  • To create an experiment template for repeated use in Diva, rick-click experiment and DUPLICATE WITHOUT DATA.
  • Export the experiment containing data (FCS files and/or experiment file) from Diva database directly to the RIA3 server
  • Delete experiments with data from Diva Database. You should have no experiments with data present in the software or on the cytometer computer.
  • If you are the last user for the day, turn off the computer.

In the event of a problem, contact Jun Case Rm. 6012C, jcase88@bwh.harvard.edu. THANK YOU!

Canto II User Guide

COMPUTER SETUP:

  • Turn on computer before turning on the machine
  • Windows 10 Username: BDOperator
  • Password: BDIS#1
  • Open Tera Term application on desktop and leave running in background

MACHINE SETUP:

  • *Prior to logging in to Diva software- Check liquid tank levels: Top off Shutdown solution tank with DI Water and Sheath tank with FACS Buffer. Before reconnecting tubing to wet cart use syringe to draw out any air bubbles in liquid lines.
  • Empty waste tank making sure to not get the filter wet. Refill with bleach to first marked line on outside of tank so that a full tank is > or = 10% solution.
  • Log in to Diva software and allow it to connect
  • Prime Tanks (Cytometer->Cleaning Modes->Prime Tank After Refill-> Check all boxes)
  • Check bubble filters on wet cart for air pockets. If air, find Flow Manager for assistance before proceeding.
    • NOTE: When you see errors of “____ laser power low” in software, it’s almost always due to air in these filters. Contact immediately.
  • Perform Fluidics Startup (Cytometer-> Fluidics Startup)
    • NOTE: only necessary for 1st user of the day
  • Give the lasers 10-15 minutes of warm-up time.

SAMPLE PREP:

  • You MUST filter samples immediately prior to loading on machine using 70uM mesh.
  • Add EDTA to prevent clumping (when possible)
  • Use a concentration that does not exceed 15k events/second when acquiring at Medium
  • Run 10% Bleach solution every 20-30 samples for 5 mins, then dH2O to prevent clogging

RUNNING SAMPLES:

  • Using two hands move the support arm to the left applying no vertical pressure to the arm and load tube on to the probe being cautious not to bend the probe. Once secured in place let arm spring back to center.
  • Begin acquiring tube using software controls and change speed of acquisition using sample speed drop down menu on acquisition dashboard.
  • For control samples, adjust voltages and settings while acquiring.
  • For experiment samples, immediately begin recording once tube is loaded to maximize the event numbers.
  • If no events, check for cracks in tube or liquid around top or on rubber fitting. Replace tube if cracked or dry and re load.
  • If still no events, possible clog has occurred. Load tube of 10% Bleach and mark meniscus with marker. Run a Clean Flow Cell (Cytometer->Cleaning Modes->Clean Flow Cell). If the liquid level does not move, repeat 2-3x. If liquid level still does not move, contact Flow Manager before proceeding. If liquid level moves, follow 2-3 clean flow cells with 2-3 more of DI Water.
  • If samples show events but are pressed up against the FSC axis, likely an air bubble. Run a Degas Flow Cell (Cytometer-> Cleaning Modes-> Degas Flow Cell)
  • Run samples at a maximum event rate of 15-20k per second.

SHUTTING DOWN:

  • After the last sample, run 10% Bleach for 5 mins and dH2O for 5 mins
  • If you are the last person on the machine, or there is no user on the machine for hours perform Fluidics Shutdown making sure the Shutdown Solution container is full of DI Water before doing so (Cytometer-> Fluidics Shutdown)
  • To create an experiment template for repeated use in Diva, right-click the experiment and DUPLICATE WITHOUT DATA.
  • Export the experiment containing data (FCS files and/or experiment file) from Diva database directly to the RIA3 server
  • Delete experiments with data from Diva Database and D: Drive once transferred to RIA3 server. You should have no experiments with data present in the software or on the computer itself.
  • Power down machine or leave on for next user.
  • Turn off the computer if you are the last user.

In the event of a problem, contact Jun Case Rm. 6012C, jcase88@bwh.harvard.edu  THANK YOU!

Troubleshooting

Computer Lag, Multiple Errors in Diva, or when in doubt…

  • Cycle power on the computer and cytometer:

    1. Power down the cytometer
    2. Shutdown the computer (not restart)
    3. Let sit for 30 seconds
    4. Turn on the computer first and allow for a full boot up and log in to Windows with PW: BDIS
    5. Open Tera Term (Fortessa) or Cytometer Status (CantoII)
    6. Power on the cytometer
    7. Open Diva software and allow it to connect
    8. If this does not fix problem, contact Core Manager

Air in flow cell or tubing

  • Fortessa:
    1. Place tube of water on probe and place cytometer in STANDBY
    2. Make sure tank next to computer has liquid in it and wet cart has full tanks and no alarm light on (RESET button on wetcart if so)
    3. Bleed air filter and line above filter on line going in to side of machine
    4. Move the sample arm to the left and REMOVE water tube
    5. PRIME 2x with no tube loaded and arm to the side
    6. RUN water tube for 1 min on HIGH
    7. Try sample tube again
    8. Repeat above steps 2-3x, if this does not fix issue contact Core Manager
  • Canto II:
    1. Check that no air is in the air filters on the wetcart (if so, contact Core Manager)
    2. Click Cytometer Menu->Cleaning Modes->Prime Tank After Refill->Check all 3 boxes->OK
    3. Click Cytometer Menu->Cleaning Modes->Bubble Filter Purge & Degas Flow Cell
    4. Repeat Step 3, 2-3x
    5. Run samples
    6. If not fixed, contact Core Manager

Clog

*users must filter all samples immediately before running on cytometer using 70um mesh filter or less

  • Fortessa
    1. Load a tube of 10% Bleach and mark meniscus
    2. Run for 2-5mins on HIGH
    3. If no liquid movement, unload tube PRIME2x, and reload tube repeating step 2.
    4. If no movement, contact Core Manager
    5. If liquid level drops and clears the clog run water for 2 mins and wipe down probe with water
  • CantoII
    1. Load tube of 10% Bleach and mark meniscus
    2. Click Cytometer Menu->Cleaning Modes->Clean Flow Cell
    3. Repeat step 2, 2-3x
    4. If no liquid movement, load tube of Bleach and run for 5mins on high
    5. If still no movement, contact Core Manager
    6. If liquid level drops, clean flow cell 2-3x with water and then run water for an additional 2 mins and wipe down the probe

Compensation

  1. Set up single-stain controls for every color in your panel
  2. Before recording samples, optimize voltages using Unstained Control worksheet to see each colors histogram and the respective spillover in to each different channel from the single control at the same time
  3. Load each single stain and make sure that the positive signal is highest in the respective channel (above 103 and ideally around 104) and does not overlap with another channel more than 50%
  4. Once each single stained control voltage is optimized to maximize it’s output and minimize overlap, record each tube and calculate compensation
  5. NOTE: Make sure that when recording, the voltages for each single-stain control are the same throughout the experiment. It will give an error message if different and not allow compensation to be calculated.

Data Management

  • Experiments containing data are not allowed to be stored within the Diva software or on the cytometer computer. They must be stored on the RIA3 server.
    1. Export all experiments containing data to RIA3/RII1 servers immediately after run
    2. If you’d like to store a blank template in the software for future use: Right-click the experiment and “Duplicate Without Data”
    3. Delete experiment containing data and save template for future experiments
  • When exporting, export data as both an “Experiment” to backup Diva software settings and as “FCS Files” for analysis using 3rd party software on your personal computer
  • For access to the RIA3/RII1 servers on the cytometer computer or your own computer, follow detailed steps found in the menu titled: RIA3/RII1 Data Storage Server Access Guide
  • WITHOUT FULL COOPERATION WE RISK COMPUTER CRASHES WHICH WIPE OUT ALL DATA AND CAUSE LENGTHY DOWN TIME OF INSTRUMENTS

RIA3/RII1 Data Storage Server Access Guide

Accessing RIA3/RII1 from FACS Computers-UPDATED 08/06/21

Email Howard Katz for ACI members or I-Cheng Ho for RII members to request access to RIA3.

Once you have access: On the Windows desktop of a FACS computer, double-click the icon labeled BWH-RIA3 (if it’s missing, see instructions* below)

A login box should pop up.  Enter your Partners user name in this format: partners\username, and enter your Partners password.  (NOTE: If a folder opens rather than a password box, and it’s not your lab’s folder, see instructions** below to change to your lab’s folder, or just use the folder named “FACS Data” to transfer your data.  The FACS Data folder can be accessed by all users of RIA3.)

Click OK

Your lab’s folder should pop up in a window.  Copy your files from the FACS computer to RIA3 as you would between any two folders in Windows.

To log off your folder, right-click on the “My Computer” icon on the Windows desktop.

Select “Disconnect Network Drive”

Select \\rfawin.partners.org

Click OK.

Contact Howard or I-Cheng if it doesn’t work.

See separate instructions for how to access your RIA3 folder from other computers.

 

* If desktop BWH-ria3 icon is missing

Right-click the “My Computer” icon on the desktop or in the Start Menu

Click “Map network drive” to open the dialog box

In the “Drive” field, select drive “Z” from the drop-down menu

In the “Folder” field, enter this address: \\rfawin.partners.org\BWH-RIA3

Be sure that “Reconnect at logon” is checked

Click on “Connect using a different user name”

Enter your Partners user name in this format: partners\username (note that it’s a back slash, not a forward slash), and enter your Partners password

Click OK

Click “Finish”

Double-click the “My Computer” icon, and you should be able to right-click on the letter of the drive you just mapped (i.e., “Z”)

Select “Create shortcut”

Click yes to the request to put it on the desktop

The RIA3-bwh icon will appear on the desktop.  You can shorten the long name Windows will put under it by right-clicking on the name and shortening it to RIA3-bwh.

 

**If RIA3 is logged into someone else’s folder

Right-click on the “My Computer” icon on the Windows desktop, or in the Start Menu

Select “Disconnect Network Drive”

Select \\rfawin.partners.org

Click OK

Log in to your lab’s folder as described at the beginning of this document, but if it still shows someone else’s folder, you should see a folder named “FACS Data.” You can copy your data to that folder, and you will be able to access the folder when you log into RIA3 from any other computer.

 

Mapping the RIA3, ACI1, and RII1 Research File Servers to Computers-UPDATED 8/6/21

Email Howard Katz for ACI members or I-Cheng Ho for RII members to request access to the appropriate servers.

Once you have access:

Windows

Right-click the “My Computer” icon on the desktop or in the Start Menu

Left-click “Map network drive” to open the dialog box

In the “Drive” field, select an unused drive letter from the drop-down menu

In the “Folder” field, copy and paste one of these addresses: \\rfawin.partners.org\BWH-RIA3, \\rfawin.partners.org\BWH-ACI1, or \\rfawin.partners.org\BWH-RII1, as appropriate.

Be sure that “Reconnect at logon” is checked

Click on “Connect using a different user name”

Click on “Finish”

Enter your Partners user name in this format: partners\username (note that it’s a backslash, not a forward slash), and enter your Partners password

Click OK

Click “Finish”

In “My Computer” or Windows File Manager, you should be able to select the letter of the drive you mapped and see folders with PI names and other names 

Repeat the process for each server you want to connect to.

Contact Howard (ACI) or I-Cheng (RII) if it doesn’t work.

 

Mac

Click on the “Go” menu

Click “Connect to Server”

In the “Server address” field, copy and paste one of these addresses: smb://rfawin.partners.org/BWH-RIA3, smb://rfawin.partners.org/BWH-ACI1, or smb://rfawin.partners.org/BWH-RII1, as appropriate.

Click “Connect”

If it asks for a login, enter your Partners user name in this format: partners\username (note that it’s a backslash, not a forward slash), and enter your Partners password

You should be able to see folders with PI names and other names. Access you PI’s lab folder to save your files. 

Repeat the process for each server you want to connect to.

Contact Howard (ACI) or I-Cheng (RII) if it doesn’t work.

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