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FCRC JumpStart

 

The Flow Cytometry Resource Center (FCRC) provides the Rockefeller University investigators with equipment and support for cell sorting (separation), acquisition, and analysis of flow cytometric and image cytometric data.

In order to use the FCRC services, all users have to fill the “PPMS account creation form” to get registered at PPMS for the Flow Cytometry Resource Center (FCRC-PPMS System). PPMS account allows users to book time on the Analyzers, Analysis-computers, Cell Sorters and ImageStream-X from the FCRC-PPMS System.

Starting from November 1st, 2019, all the FCRC Class, Consultation, Training and Help Request Forms have been transferred to the FCRC-PPMS System. This is in addition to the regular booking and usage of the Cell Sorters, ImageStream-X, Analyzers and Analysis-computers. Detailed information please click here.

You also need to have CFS (RU Central File Storage) login/password in order to transfer data from and to the Analyzers and Analysis-computers, as well as retrieving data from the Cell Sorters. To learn more about the CFS please refer to the RU IT Website at http://it.rockefeller.edu/file-storage.


FCRC training_noMA900   

BD Accuri C6


SORTERS

Cell sorting is performed either by FCRC staff (BD FACSAria) or by trained users (Sony MA900, Coming Soon). The three BD FACSAria cell sorters equipped with up to six lasers (488, 561, 640, 355, 405, and 445 nm excitation wavelengths) and 18 fluorescence channel detection, while Sony MA900 is with four lasers (488, 405, 561 and 640 nm excitation wavelengths) and 12 fluorescence channels detection. All the cell sorters are able to perform high-speed (FACSAria up to 20,000 events/sec and MA900 up to 70,000 events/sec), high-purity (up to 99.9%) sterile sorts into different types of devices (tubes, plates, slides, or custom-made devices). BD FACSAria sorters are operated by FCRC staff during the extended business hours. For exact time, please refer to the Cell Sorting and ImageStream-X Schedule. Sony MA900 (Coming Soon) can be operated by researchers with full time (24/7/365) access, after completion of the required training.

 

 
 
 
 
BD FACSAria

Notes:

  • All BD FACSAria sorters at FCRC are equipped with the bioBUBBLE Benchtop Biocontainment Enclosures, which allow the sorters to be dedicated to the sort of the RG2 materials due to the compliance with the BSL-2+ requirements
  • BD FACSAria-3 is the only sorter at FCRC dedicated to the sort of the CFP-expressing cells due to the presence of the 445 nm laser

In order to start using the Cell Sorters, users need to:

  • Create FCRC-PPMS account by Filling the “PPMS account creation form” to get registered at PPMS for the Flow Cytometry Resource Center (FCRC-PPMS System)
  • Study Virtual Classroom for Pre-Sort Consultation
  • The “Beyond the Basics” Flow Cytometry Class (FCRC-PPMS Request Form A1) is strongly suggested
  • Participate in the Experimental Design Consultation (FCRC-PPMS Request Form C1) if needed
  • Participate in the Pre-Sort Consultation
    1. Submit the Pre-Sort Consultation Form (FCRC-PPMS Request Form B1)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Attend the consultation. Bring (or have available) all information related to the planned experiment (the samples are not needed)

To book the FACSAria Sort, users need to:

  • Login to FCRC-PPMS System and follow the directions https://fcrcppms.rockefeller.edu/vdoc/?pf=2&docid=3
  • Receive the follow-up confirmation email from FCRC with the date/time/duration for the Cell Sort originally requested
  • On the date/time of the scheduled Cell Sort come to FCRC Sorting Room (DWB207) with controls (compensations and FMO), titrations, and samples with the concentrations suggested at the Pre-Sort Consultation, as well as all buffers, tubes, plates

Notes:

  • If you cannot make the sorting appointment on time, please make sure to notify about the delay with the start of the sort by calling #7657
  • Despite the possible delay with the start time, the end time for the sort by default remains unchanged. Please plan accordingly
  • In some cases the extension of the sorting time is possible (schedule permitted), but needs to be discussed with the FCRC Director

To cancel the Cell Sort, users need to:

  • Login to FCRC-PPMS System and cancel your sort
  • Receive the follow-up confirmation email from FCRC

Note: Please refer to the FCRC CHARGES AND CANCELLATION POLICY

To reschedule the existing Cell Sort, users need to:

  • Login to FCRC-PPMS System
  • Cancel your sort
  • Receive the follow-up confirmation email from FCRC
  • Book new sort
  • Receive the follow-up confirmation email from FCRC with the date/time/duration of the new scheduled Cell Sort
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Sony MA900

Coming soon…

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ANALYZERS

The FCRC is equipped with seven benchtop analyzers, which cover the wide range of researcher’s needs: The Image Analyzer – The Amnis ImageStream-X combines the strength of flow cytometry and fluorescence microscopy in a single platform. It allows for high content assays on rare cells and quantification of biological phenomena with incredible accuracy. The Spectral Analyzer – Cytek Aurora, has five lasers (355, 405, 488, 561 and 640 nm excitation wavelengths) and 64 fluorescence channels that can measure the entire emission spectra of the fluorescent dyes excited by multiple lasers installed on the instrument. Till now maximum 40 different fluorescent labels could be resolved, even when multiple markers are co-expressed on the same cell. Four Advanced benchtop Analyzers: Three from BD – two BD LSRIIs and one BD LSR-Fortessa, have up to five lasers (488, 561, 640, 355 or 445 and 405 nm excitation wavelengths) and 18 fluorescence channels; One from ThermoFisher –  ThermoFisher Attune NxT is equipped with two lasers (488 and 561 nm excitation wavelengths) and seven fluorescence channels which could run samples in tubes or in plate formats, including 96-well, 384-well, and deep-wells with the high volume intake. The Center also has a Basic Analyzer:  BD Accuri® C6 has two lasers (488 and 640 nm excitation wavelengths) and four fluorescence channels.

 

 
 
 
 
AMNIS IMAGESTREAM-X

The ImageStream-X is able to simultaneously record up to 10-color images with the four-laser excitation (488, 561, 658, and 407 nm wavelengths). Data acquisition on ImageStream-X is performed by FCRC staff during the business hours. For exact times, please refer to the Cell Sorting and ImageStream-X Schedule.

In order to start use the ImageStream-X, users need to:

  • Create FCRC-PPMS account by Filling the “PPMS account creation form” to get registered at PPMS for the Flow Cytometry Resource Center (FCRC-PPMS System)
  • Activate RU card for DWB406 FCRC door access (FCRC-PPMS Request Form A0)
  • Study Virtual Classroom is strongly suggested
  • The “Beyond the Basics” Flow Cytometry Class (FCRC-PPMS Request Form A1) is strongly suggested
  • Participate in the Pre-ImageStream Consultation
    1. Submit the Pre-ImageStream Consultation From (FCRC-PPMS Request Form C2)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Attend the consultation. Bring (or have available) all information related to the planned experiment

To book the ImageStream-X, users need to:

  • Login to FCRC-PPMS System and follow the directions https://fcrcppms.rockefeller.edu/vdoc/?pf=2&docid=3
  • Receive the follow-up confirmation email from FCRC with the date/time/duration for the ImageStream Acquisition originally requested
  • On the date/time of the scheduled ImageStream Acquisition come to FCRC Room (DWB406) with all controls (unstained, single-color) and samples with the concentrations and volume suggested at the Pre-ImageStream Consultation

Notes:

  1. If you cannot make the ImageStream-X appointment on time, please make sure to notify about the delay with the start of the acquisition by calling #7657
  2. Despite the possible delay with the start time, the end time for the acquisition by default remains unchanged. Please plan accordingly
  3. In some cases the extension of the ImageStream-X acquisition time is possible (schedule permitted), but needs to be discussed with the FCRC Director

To cancel the ImageStream-X, users need to:

  • Login to FCRC-PPMS System and cancel your acquisition
  • Receive the follow-up confirmation email from FCRC

Note: Please refer to the FCRC CHARGES AND CANCELLATION POLICY<

To reschedule the existing ImageStream-X, users need to:

  • Login to FCRC-PPMS System
  • Cancel your acquisition
  • Book new acquisition
  • Receive the follow-up confirmation email from FCRC with the date/time/duration of the new scheduled ImageStream Acquisition

Note: In case the ImageStream need to be rescheduled go to FCRC-PPMS to cancel the first appointment and book the new one instead

To be able to analyze data, users need to:

  • Submit the IDEAS Software Training Form (FCRC-PPMS Request Form F1)
  • Receive confirmation email from FCRC with the date/time confirmed
  • Attend the training
  • After the IDEAS Introduction training, the FCRC users should be able to analyze their data independently
  • In case users need FCRC staff help with the Further Data Analysis, they should submit the IDEAS Software Troubleshooting Form (FCRC-PPMS Request Form F1)
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Spectral Analyzer
In order to start using the Spectral Analyzer – Cytek Aurora, users need to:

  • Create FCRC-PPMS account by Filling the “PPMS account creation form” to get registered at PPMS for the Flow Cytometry Resource Center (FCRC-PPMS System)
  • Activate RU card for DWB211 FCRC door access (FCRC-PPMS Request Form A0)
  • Study Virtual Classroom
  • Participate in the ”Beyond the Basics” Flow Cytometry Class
    1. Submit the Flow Cytometry Class Application form (FCRC-PPMS Request Form A1) for the certain date
    2. Receive confirmation email from FCRC
    3. Attend the Class
    4. Get login/password to login into SpectroFlo program on Cytek Aurora
  • Participate in the Pre-Hands-On Consultation
    1. Submit the Aurora Pre-Hands-On Consultation Form (FCRC-PPMS Request Form C4)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Attend the consultation. Bring (or have available) all information related to the planned experiments
  • Get the Cytek Aurora Hands-On Training
    1. Submit the Aurora Hands-On Training Form (FCRC-PPMS Request Form D3)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Book Cytek Aurora for the date/time suggested in the FCRC confirmation email
    4. Come to the Hands-on Training with the samples suggested at the Pre-Hands-On Consultation (usually – unstained, all single-color controls and a few samples)
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Advanced Analyzers

 
In order to start using the Advanced Analyzers – BD LSR (BD LSRIIs and BD LSR-Fortessa) and ThermoFisher Attune NxT, users need to:

  • Create FCRC-PPMS account by Filling the “PPMS account creation form” to get registered at PPMS for the Flow Cytometry Resource Center (FCRC-PPMS System)
  • Activate RU card for DWB211/406 FCRC door access (FCRC-PPMS Request Form A0)
  • Study Virtual Classroom
  • Participate in the ”Beyond the Basics” Flow Cytometry Class
    1. Submit the Flow Cytometry Class Application form (FCRC-PPMS Request Form A1) for the certain date
    2. Receive confirmation email from FCRC
    3. Attend the Class
    4. Get login/password to login into Diva program on BD LSRIIs and BD LSR-Fortessa
  • Participate in the Pre-Hands-On Consultation
    1. Submit the Pre-Hands-On Consultation Form for BD LSR (FCRC-PPMS Request Form C3) or Attune NxT (FCRC-PPMS Request Form C5)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Attend the consultation. Bring (or have available) all information related to the planned experiments
  • Get the Hands-On Training
    1. Submit the Hands-On Training Form for BD LSR (FCRC-PPMS Request Form D1) or Attune NxT (FCRC-PPMS Request Form D5)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Book BD LSRIIs and BD LSR-Fortessa or ThermoFisher Attune NxT for the date/time suggested in the FCRC confirmation email (LSRII-1 and LSR-Fortessa are equipped with 355nm laser, while LSRII-2 is equipped with 445nm laser) or ThermoFisher Attune NxT for the date/time suggested in the FCRC confirmation email
    4. Come to the Hands-on Training with the samples suggested at the Pre-Hands-On Consultation (usually – unstained, all single-color controls and a few samples)
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Basic Analyzer

In order to start using the Basic Analyzer – BD Accuri® C6, users need to:

  • Create FCRC-PPMS account by Filling the “PPMS account creation form” to get registered at PPMS for the Flow Cytometry Resource Center (FCRC-PPMS System)
  • Activate RU card for DWB211/406 FCRC door access (FCRC-PPMS Request Form A0)
  • Study Virtual Classroom
  • The “Beyond the Basics” Flow Cytometry Class (FCRC-PPMS Request Form A1) is strongly suggested
  • Participate in the Pre-Hands-On Consultation
    1. Submit the BD Accuri C6 Pre-Hands-On Consultation Form (FCRC-PPMS Request Form C6)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Attend the consultation. Bring (or have available) all information related to the planned experiments
  • Get the BD Accuri® C6 Hands-On Training
    1. Submit the BD Accuri C6 Hands-On Training Form (FCRC-PPMS Request Form D6)
    2. Receive confirmation email from FCRC with the date/time specified
    3. Book BD Accuri C6 for the date/time suggested in the FCRC confirmation email
    4. Come to the Hands-on Training with the samples suggested at the Pre-Hands-On Consultation (usually – unstained, all single-color controls and a few samples)

 

After completion of these steps, researchers have full time (24/7/365) access to the analyzers and can operate the analyzers themselves. In case of essential troubleshooting or help need from FCRC staff, please make sure to fill the Instrument Help Form (FCRC-PPMS Request Form E1-E5)

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STAFF ASSISTANCE

The FCRC staff members are available for assisted operation of the Analyzers, assisted data analysis, consultation, instrument and software training and troubleshooting from 9:00am – 6:00pm weekdays. Please send the request form for your specific consultation, training and help need.

  1. Consultations
    • Experimental Design Consultation (FCRC-PPMS Request Form C1)
    • Pre-Sort Consultation (FCRC-PPMS Request Form B1)
    • Pre-ImageStream Consultation (FCRC-PPMS Request Form C2)
    • Pre-Hands-On Consultation (FCRC-PPMS Request Form C3-C6)
  2. Instrument Hands-On Training
    • Cytek Aurora Hands-on Training (FCRC-PPMS Request Form D3)
    • Cytek Aurora AMS Hands-on Training (FCRC-PPMS Request Form D4)
    • BD LSRIIs and BD LSR-Fortessa Hands-on Training (FCRC-PPMS Request Form D1)
    • BD LSRII HTS Hands-on Training (FCRC-PPMS Request Form D2)
    • ThermoFisher Attune NxT Hands-on Training (FCRC-PPMS Request Form D5)
    • BD Accuri C6 Hands-on-Training (FCRC-PPMS Request Form D6)
  3. Instrument Help (FCRC-PPMS Request Form E1-E5)
    • Help with Instrumental Setup
    • Troubleshooting on the Instrument
  4. Software Troubleshooting
    • IDEAS (FCRC-PPMS Request Form F1)
    • SpectroFlo (FCRC-PPMS Request Form F2)
    • FCS Express (FCRC-PPMS Request Form F3)
    • FlowJo (FCRC-PPMS Request Form F4)
    • CSampler (FCRC-PPMS Request Form F5)

Note: In case of emergency or urgent quick questions on the instrument, please do not hesitate to contact FCRC at any time, stop by FCRC (DWB205) on the weekdays (9:00am-6:00pm), or send email to FCRC staff during weekends and off hours.


ACKNOWLEDGEMENTS

  • Please remember to acknowledge the Flow Cytometry Resource Center (FCRC) staff in publications. Co-authorship on publications for Resource Center staff may be considered appropriate when the staff have provided one or more of the following:
    • Significant intellectual contribution to the design of the published experiments;
    • Substantial practical contribution to the generation, analysis and/or interpretation of experimental data;
    • Indispensable technical support that contributes intellectually or scientifically to the advancement of the work.

Resource Center staff members who have made an intellectual or technical contribution, but of a limited nature and not justifying authorship as defined above, should be given an appropriate acknowledgement in any resulting publications. This will enable us to demonstrate our involvement in the research of a large number of laboratories at the university, aiding the procurement of future equipment.

  • Condition of NYSTEM funding for the FACSAria II-2
    • Support through the NYSTEM grant shall be acknowledged in all publications, presentations and products of research resulting from use of or data acquired through the BD FACSAriaII-2, in a form consistent with the publication’s guidelines
    • The acknowledgement should reference “Support by the Empire State Stem Cell fund through NYSDOH Contract #C023046” and include following statement “Opinions expressed here are solely those of the author and do not necessarily reflect those of the Empire State Stem Cell Fund, the NYSDOH, or the State of NY”
    • Please be sure to send to FCRC_Staff@rockefeller.edu documentation for publications funded wholly or in part by the contract that include an acknowledgment of this funding support. This information must be submitted to NYSTEM within 60 days of publication

For More information, please refer to Guidelines!

SEE YOU SOON IN FCRC!