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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.

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.

ANALYZERS

The FCRC is equipped with six benchtop analyzers, which cover the wide range of researcher’s needs: The Spectral Analyzer — Cytek Aurora, the three laser (488, 640 and 405 nm excitation wavelengths) and 38 fluorescence channels that can measure the entire emission spectra of the fluorescent dyes excited by multiple lasers installed on the instrument. At least 23 different fluorescent labels could be resolved, even when multiple markers are co-expressed on the same cell. Three Advanced benchtop Analyzers — 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. The Center also has two Basic Analyzers: 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. BD Accuri® C6 with two lasers (488 an 640 nm excitation wavelengths) and four fluorescence channels.

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

  • Study Virtual Classroom
  • Activate RU card for DWB211 FCRC door access
  • Participate in the Pre-Hands-On Consultation
    1. 1. Submit the Pre-Hands-On Consultation Form
    2. 2. Receive confirmation email from FCRC with the date/time specified
    3. 3. Attend the consultation. Bring (or have available) all information related to the planned experiments
  • Get the ThermoFisher Attune NxT or BD Accuri® C6 Hands-On Training
    1. 1. Submit the ThermoFisher Attune NxT or BD Accuri® C6 Hands-On Training Form
    2. 2. Receive confirmation email from FCRC with the date/time specified
    3. 3. Book ThermoFisher Attune NxT or BD Accuri® C6 for the date/time suggested in the FCRC confirmation email
    4. 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)

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

  • Study Virtual Classroom
  • Activate RU card for DWB211 FCRC door access
  • Participate in the ”Beyond the Basics” Flow Cytometry Class
    1. 1. Submit the Flow Cytometry Class Application form for the certain date
    2. 2. Receive confirmation email from FCRC
    3. 3. Attend the Class
    4. 4. Get Diva login/password to login into Diva program on BD LSRIIs and BD LSR-Fortessa
  • Participate in the Pre-Hands-On Consultation
    1. 1. Submit the Pre-Hands-On Consultation Form
    2. 2. Receive confirmation email from FCRC with the date/time specified
    3. 3. Attend the consultation. Bring (or have available) all information related to the planned experiments
  • Get the BD LSRs and BD LSR-Fortessa Hands-On Training
    1. 1. Submit the BD LSR Hands-On Training Form
    2. 2. Receive confirmation email from FCRC with the date/time specified
    3. 3. Book BD LSRIIs and BD LSR-Fortessa 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)
    4. 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)

In order to start using the Spectral Analyzers – Cytek Aurora, users need to:

  • Study Virtual Classroom
  • Activate RU card for DWB211 FCRC door access
  • Participate in the ”Beyond the Basics” Flow Cytometry Class
    1. 1. Submit the Flow Cytometry Class Application form for the certain date
    2. 2. Receive confirmation email from FCRC
    3. 3. Attend the Class
    4. 4. Get Diva login/password to login into Diva program on Cytek Aurora
  • Participate in the Pre-Hands-On Consultation
    1. 1. Submit the Pre-Hands-On Consultation Form
    2. 2. Receive confirmation email from FCRC with the date/time specified
    3. 3. Attend the consultation. Bring (or have available) all information related to the planned experiments
  • Get the Cytek Aurora Hands-On Training
    1. 1. Submit the Cytek Aurora Hands-On Training Form
    2. 2. Receive confirmation email from FCRC with the date/time specified
    3. 3. Book Cytek Aurora for the date/time suggested in the FCRC confirmation email
    4. 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.


SORTERS

Cell sorting is performed by FCRC staff on 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. All the cell sorters are able to perform high-speed (up to 20,000 events/sec), high-purity (up to 99.9%) sterile sorts into different types of devices (tubes, plates, slides, or custom-made devices). Cell sorters are operated by FCRC staff during the extended business hours. For exact time, please refer to the Cell Sorting and ImageStream-X Schedule.

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:

To book the Cell 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:

  • 1. 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
  • 2. Despite the possible delay with the start time, the end time for the sort by default remains unchanged. Please plan accordingly
  • 3. 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

IMAGESTREAM-X

The 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 ImageStream-X is able to simultaneously record up to 10-color images with the four-laser excitation (488, 561, 658, and 405 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:

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 (DWB211) with all controls (unstained, single-color) and samples with the concentrations and volume suggested at the Pre-ImageStream Consultation

Notes:

  1. 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. 2. Despite the possible delay with the start time, the end time for the acquisition by default remains unchanged. Please plan accordingly
  3. 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 (Select option: Introduction)
  • 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

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. 1. Consultations
    • Experimental Design Consultation
    • Pre-Sort Consultation
    • Pre-ImageStream Consultation
    • Pre-Hands-On Consultation
  2. 2. Instrument Hands-On Training
    • Cytek Aurora hands-on Training
    • BD LSRIIs and BD LSR-Fortessa hands-on Training
    • HTS Hands-on Training
    • ThermoFisher Attune NxT hands-on Training
    • BD Accuri® C6 Hands-on-Training
  3. 3. Instrument Help
    • Help with Instrumental Setup
    • Troubleshooting on the Instrument
  4. 4. Software Training (Introduction or Troubleshooting)
    • IDEAS
    • FlowJo
    • FCS Express
    • CSampler
    • FCAP Array
  5. 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 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@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!