Progress 09/01/23 to 08/31/24
Outputs
Target Audience: The target audience for use of the Attune CytPix, based upon the research areas proposed within the EGP, include agricultural and environmental researchers. In addition, we have trained users that have projects that focus on human health and therapeutic development for diseases such as cancer. Changes/Problems:1) During the 1st year of use, we encoutered a number of issues concerning the operation of the instrument. The original CytPix experienced a number of hardware related critical failures during the first year (e.g., 488nm laser replacement, 3 rotary valve replacements, 2 flow cell replacements, autosampler replacement). As these failures were well outside the manufacturer's specifications for failures, and the instrument manufacturer was unable to identify a root cause for these failures related to the instrument use, this precipitated discussions and negotiations with ThermoFisher that ultimately led to the manufacturer supporting replacement of the original CytPix with a branrd new Attune CytPix. The new instrument was swapped out and installed in January 2024 at no additional cost to the Core or MSU. Since the installation the operational issues have been resolved. 2) We initially established a monthly 'User Group,' the goal of which was to discuss results and share troubleshooting experiences. These meetings were initiated in 2023. As we noticed that the in-person attendance for these monthly meetings was progressively declining, possibly due to the overall frequency, we began offering these in an intermittent format. Since changing the format to hybrid or all virtual we have observed increased interest and attendance. We anticipate that we will support approximately 6 relevant 'User Group'-associated topics per year, effectively modifying this goal to a bi-monthly (every other) frequency. What opportunities for training and professional development has the project provided?Core Staff: Incorporation of the Attune CytPix has provided developmental opportunities to Core staff by increasing the number of collaborations we have built over the past 1.5 years. Further, increasing image processing capabilities with Attune software updates has provided an opportunity for staff to expand our knowledge of image analysis (both on the Attune software and FCS Express) that will benefit current and future users. A number of anticipated software updates will allow us to perform high-dimensional reduction and clustering analyses on imaging parameters in the Attune CytPix software, making it easier to identify parameters that make different clusters unique. Dr. Daniel Vocelle (Assistant Director) serves as a member of the ThermoFisher Scientific Advisory Board for Cytometry, at the request of the company. This relationship helps us drive attention to future instrumentation and software developments for the CytPix. Trainees/Users: Benchtop analyzer capabilities in the MSU Flow Cytometry Core Facility, including sample collection automation (autosampler), yellow-green laser excitation, and brightfield imaging capabilities increases developmental opportunities for trainees to learn new acquisition options and imaging processing that can enhance data generated for their projects. How have the results been disseminated to communities of interest?Currently, we are aware of a minimum of 4 accepted and published manuscripts that have utilized the Attune CytPix. Three of these publications come from 2 major user labs whose projects were described in the EGP. One of these publications includes Dr. Matthew Bernard (PD; Director) as a co-author for contribution to assay development. https://doi.org/10.1128/mbio.02043-23 https://doi.org/10.1128/iai.00060-23 https://doi.org/10.1186/s40104-024-00999-5 https://doi.org/10.4049/immunohorizons.2300107 Dr. Daniel Vocelle (Assistant Director; primary author) and Dr. Matthew Bernard (PD; Director, co-author) have developed a project and novel use of the brightfield-imaging capabilities of the Attune CytPix with one of the major users described in the EGP (Dr. Nina Wale). This publication is currently on BioRIV and expected to be peer-reviewed soon. https://doi.org/10.1101/2024.05.02.592280 Dr. Vocelle has also presented this work in multiple formats, including a number of invited talks listed below. Poster: Phenotyping and Sorting Pathogens Based on Distinct Autofluorescent and Morphological Parameters CYTO 2024 *International), May 2024, Edinburgh Scotland. Invited Talk: "Label-free cell sorting of distinct microbial populations based on autofluorescent and morphological phenotypes derived from imaging flow cytometry." FlowTex Annual Conference 2024, March 2024, Houston, TX. Invited Talk: "Exploiting Autofluorescence and Imaging Flow Cytometry to Quantitatively Phenotype Morphologically Diverse Pathogen Populations." Frontiers In Flow Cytometry™ 2023 (International), September 2023, Virtual. Invited Talk: "The Shape of Sickness: Connecting Bacterial Morphology to Virulence Using Image-based Flow Cytometry." GLIIFCA Annual Conference 2023, September 2023, Madison, WI. Investigators have pledged to continue to appropriately include USDA NIFA EGP grant reference in the Acknowledgements section of any publications utilizing the Attune CytPix. What do you plan to do during the next reporting period to accomplish the goals?Staff outreach to a diverse selection of departmental faculty meetings, student research forums, individual labs/organizations, etc. within MSU and externally to promote instrumentation and Core capabilities will continue in order to grow the user base for the CytPix. Additionally, Dr. Bernard and Vocelle, will continue to collaborate with labs to develop novel uses for the Attune CytPix, the goal of which will be to provide research grant support, future publications, and dissemination of cutting-edge research results. Additionally, we will continue to develop relevant 'User Group' meetings and bring new applications and technologies to our Attune CytPix users.
Impacts
What was accomplished under these goals?
1) All sub-goals for Major Goal #1 have been addressed and successfully completed, and the instrument has been fully integrated into the Core. The Attune CytPix was purchased, delivered, and installed in the MSU Flow Cytometry Core Facility (BPS Room 4120). Initial installation date occurred on November 3-4, 2022. As adjustments were required by the vendor after the initial install, the final official installation date was December 1, 2022, which is what was considered the start of 1-year maintenance contract with ThermoFisher. MFCCF staff received vendor-led training on instrument operation and maintenance on November 29-30, 2022. The fully integrated instrument was available for use at no charge from December 2022 to November 30, 2023. A University-approved billing rate ($39/hr) took effect on December 1, 2023. The BD LSR II was officially retired in 2024 and billing rates were terminated for this service on 05/01/2024. 2) All sub-goals for Major Goal #2 have been addressed. We continue to develop appropriate virtual and hands-on training, as instrument software upgrades occur. A quick guide, outlining standard operating procedures and best practices has been developed and implemented. It is made available to all users in our Google Classroom and is laminated with the Attune CytPix for immediate access while using the instrument. We have developed an on-demand virtual training module for basic Attune CytPix training. This has been made available to all trainees in our Google Classroom that we use to provide all asynchronous training to users. Additionally, as more advanced image processing capabilities become available with each software update, we are updating analysis training provided to users. We developed and implemented a hands-on training program for users of the Attune CytPix and training began in December of 2022. This consists of 4 training sessions. The first training session uses beads provided by the Core to demonstrate instrument and software operation. The second training allows users to bring positive and negative controls to learn to optimize instrument settings with their samples. The third training session allows users to bring a set of experimental samples with appropriate controls to further gain confidence using the instrument to support their research project. The fourth training session focuses on data analysis of the data generated from training sessions 2 and 3. We had 950+ hours of use on the Attune CytPix in the first year from installation to billing rate institution prior to December 1, 2023 (average monthly use in Dec. 2022-Nov. 2023 = 83 hours/month). Following billing rate implementation on Dec. 1, 2023, the average monthly use has been 90 hours/month (Dec. 2023. - May 2024). As of today (06/25/2024), we have on-boarded and trained 79 unique users from 39 different labs on the Attune CytPix, who are now able to independently operate the instrument. Of the 10 major user labs/projects that were described in the original EGP we have trained users from 9 out of 10 of those "major user" labs. 3) All sub-goals for Major Goal #3 have been addressed with some modifications to the frequency of 'User Group' meetings. We initially established a monthly 'User Group,' the goal of which is to discuss results and share troubleshooting experiences. These meetings were initiated in 2023. Highlights included a Thermo Scientific sponsored overview of the Attune capabilities; BioLegend sponsored presentation on flow cytometry reagents and best practices; a ThermoFisher sponsored training for a CytPix software update that has provided enhanced image processing data; A Core-led presentation by Dr. Daniel Vocelle (Assistant Director) outlining how to utilize new imagining parameters (20 new parameters measured by image processing) with high dimensional reduction and clustering analysis. As we noticed that the in-person attendance for these monthly meetings was progressively declining, possibly due to the overall frequency, we began offering these in an intermittent format. Since changing the format to hybrid or all virtual we have observed increased interest and attendance. Highlights include: a ThermoFisher-led discussion of ROS detection by flow cytometry; DeNovo software led a cell cycle analysis training session in FCS Express (data analysis software); Tissue dissociation seminar led by Miltenyi Biotec to discuss better prep of samples for flow cytometry; DeNovo software led a imaging analysis training session in FCS Express; exploration ability to sort cells based on imaging parameters (ThinkCyte webinar). We anticipate that we will support approximately 6 relevant 'User Group'-associated topics per year, effectively modifying this goal to a bi-monthly frequency. Both Dr. Matthew Bernard (PD, Director) and Dr. Daniel Vocelle (Assistant Director) presented Core instrumentation (including the CytPix) and capabilities at ~12 different departmental faculty meetings; departmental retreats, symposiums in 2023 and 2024. We will continue to target any appropriate forums to promote capabilities and instrumentation. Both Dr. Matthew Bernard (PD, Director) and Dr. Daniel Vocelle (Assistant Director) continually reach out to non-Flow Cytometry Core user labs individually to promote the technology and will continue to do so to build up a user base on the instrument. Additionally, we have built up relationships with 20+ departments and frequently meet with new faculty recruits on their 2nd visit to discuss capabilities of the Core, including the Attune CytPix. Dr. Daniel Vocelle (Assistant Director) has presented data in several invited talks showing a novel use of the Attune CytPix: Poster: Phenotyping and Sorting Pathogens Based on Distinct Autofluorescent and Morphological Parameters CYTO 2024 *International), May 2024, Edinburgh Scotland. Invited Talk: "Label-free cell sorting of distinct microbial populations based on autofluorescent and morphological phenotypes derived from imaging flow cytometry." FlowTex Annual Conference 2024, March 2024, Houston, TX. Invited Talk: "Exploiting Autofluorescence and Imaging Flow Cytometry to Quantitatively Phenotype Morphologically Diverse Pathogen Populations." Frontiers In Flow Cytometry™ 2023 (International), September 2023, Virtual. Invited Talk: "The Shape of Sickness: Connecting Bacterial Morphology to Virulence Using Image-based Flow Cytometry." GLIIFCA Annual Conference 2023, September 2023, Madison, WI. Investigators have pledged to continue to appropriately include USDA NIFA EGP grant reference in the Acknowledgements section of any publications utilizing the Attune CytPix.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
https://doi.org/10.1128/mbio.02043-23
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
https://doi.org/10.1128/iai.00060-23
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
https://doi.org/10.1186/s40104-024-00999-5
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
https://doi.org/10.4049/immunohorizons.2300107
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The target audience for use of the Attune CytPix, based upon the research areas proposed within the EGP, include agricultural and environmental researchers. In addition, we have trained users that have projects that focus on human health and therapeutic development for diseases such as cancer. Changes/Problems:It is likely that the LSR II will not be fully retired until at least 2024, in order to give additional time to labs that have ongoing projects that require its continued use. Overall, there is no major disadvantage to keeping the LSR II in operation as cost to maintain is now much lower, as instrument is no longer on contract and significantly reduced use decreases the cost for consumables and reduces repair/maintenance frequency. What opportunities for training and professional development has the project provided?Core Staff: Incorporation of the Attune CytPix has provided developmental opportunities to Core staff by increasing the number of collaborations we have built in the past 6 months. Further, increasing image processing capabilities has provided an opportunity for staff to expand our knowledge of image analysis (both on the Attune software and FCS Express) that will benefit current and future users. Trainees/Users: New benchtop analyzer capabilities in the MSU Flow Cytometry Core Facility, including sample collection automation (autosampler), yellow-green laser excitation, and brightfield imaging capabilities increases developmental opportunities for trainees to learn new acquisition options and imaging processing that can enhance data generated for their projects. How have the results been disseminated to communities of interest?Currently, we have several labs that have generated data using the Attune CytPix that is intended to be included in publications. Investigators have pledged to appropriately include USDA NIFA EGP grant reference in the Acknowledgements section of any publications utilizing the Attune CytPix. Additionally, Dr. Daniel Vocelle (Assistant Director) was invited by Thermo Fisher discuss our experience with the Attune and highlights the unique capability that is brings to MSU investigators. This invited presentation is titled: Benefits of Imaging Data in Flow Cytometry Experiments: A Panel Discussion" that occurred on June 29th, 2023. What do you plan to do during the next reporting period to accomplish the goals?We will continue to develop our monthly 'User Group' meetings. Once a significant cohort of users have generated data across a variety of projects, we will implement user/trainee-led discussions focusing on successes, unique data sets, and troubleshooting. Staff outreach to a diverse selection of departmental faculty meetings, student research forums, individual labs/organizations, etc. within MSU and externally to promote instrumentation and Core capabilities will continue in order to grow the user base for the CytPix.
Impacts
What was accomplished under these goals?
1) All sub-goals for Major Goal #1 have been at least partially addressed or successfully completed, and the instrument has been fully integrated into the Core. The Attune CytPix was purchased, delivered, and installed in the MSU Flow Cytometry Core Facility (BPS Room 4120). Initial installation date occurred on November 3-4, 2022. As adjustments were required by the vendor after the initial install, the final official installation date was December 1, 2022, which is what is considered the start of 1-year maintenance contract with Thermo Fisher. MFCCF staff received vendor-led training on instrument operation and maintenance on November 29-30, 2022. The fully integrated instrument is currently free for Core users. A University-approved billing rate ($39/hr) was submitted and approved and will take effect on December 1, 2023. The BD LSR II was moved to BPS Room 4120 in the Spring of 2023. All major users that did not have ongoing projects requiring use of the LSR II. As of 6/15/2023, we have cross-trained 14 previous LSR II users. Average weekly use for the Attune over the past month was 23 hours, while average weekly use of the LSR II was 2.9. Previous average weekly LSR II use was approximately 13.7 hours, indicating that we have significantly diminished the number of use hours on the LSR II in order to allow users to utilize the cutting-edge Attune CytPix. 2) All sub-goals for Major Goal #2 have been at least partially addressed. We continue to develop appropriate virtual and hands-on training, as instrument software upgrades occur. A quick guide, outlining standard operating procedures has been developed and implemented. It is made available to all users in our Google Classroom and is laminated with the Attune CytPix for immediate access while using the instrument. We have developed an on-demand virtual training module for basic Attune CytPix training, as well as more advanced image processing and analysis. This will be made available to all trainees in July 2023. All future trainees will be required to view this material and complete an associated quiz prior to beginning hands-on training. All users that were previously trained prior to July 2023, will also be encouraged to view the materials as a refresher training. We developed and implemented a hands-on training program for users of the Attune Cytpix and training began in December of 2022 for major LSR II users. To date we have on-boarded and trained 36 users from 24 different labs on the Attune CytPix, who are now able to independently operate the instrument. 3) All sub-goals for Major Goal #3 have been addressed. We have established a monthly 'User Group,' the goal of which is to discuss results and share troubleshooting experiences. These meetings were initiated in 2023. Highlights include a Thermo Scientific sponsored overview of the Attune capabilities; BioLegend sponsored presentation on flow cytometry reagents and best practices;, a ThermoFisher sponsored training for a CytPix software update that has provided enhanced image processing data; A Core-led presentation by Dr. Daniel Vocelle (Assistant Director) outlining how to utilize new imagining parameters (20 new parameters measured by image processing) with high dimensional reduction and clustering analysis. Both Dr. Matthew Bernard (PD, Director) and Dr. Daniel Vocelle (Assistant Director) presented Core instrumentation (including the CytPix) and capabilities at ~10 different departmental faculty meetings. We will continue to target any appropriate forums to promote capabilities and instrumentation.
Publications
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