Flow cytometer: The Luminex (previously Millipore) provides 3 lasers (violet, blue, and red) and 10 fluorescence channels for the detection of fluorescence-labeled cells and particles. This flow cytometer uses microcapillary to allow direct sampling by aspiration and eliminates the need of sheath fluid. The automated sampler for a 96-well plate and 10 sample tubes enables walkaway sample handling for higher throughput assays. A rotating and variable-speed mixing paddle is used between each well to help cells stay in suspension during sampling. These features provide advantages and cause errors meanwhile in sample measurement.
Sample format Samples should be prepared with 96-well sample plates (PDF) (U-bottom preferred) or 1.5ml sample tubes (PDF). The cell suspension should be maintained at ~200 cell/ml and ~200 ul/well for the 96-well plate format.
Data Storage A project drive, called “Document-Flow users”, has been set up for temporary storage. If the computer space is limited, the stored data will be removed after 6-month from initial acquisition.
Data Analysis Data can be analyzed with the software GuavaSoft inCyte or Guava Suite provided by the manufacturer. Data can also be analyzed with FlowJo (group license is available from CCHMC flow cytometry core), FCS Express, and ModFit LT, which are also available for data analysis free of charge to CCHMC flow core users in R5503. See "Software" for more information.
Users are encouraged to understand the basic knowledge of flow cytometry in experimental design, antibody staining, compensation, and data analyses together with unique setting for this easyCyte unit to ensure success. These resources include the instrument website of Guava easyCyte 12-channel high throughput (12HT) flow cytometer, flow cytometry resources, on campus lectures provided by Ohio River Valley Cytometry Association (ORVCA), and related seminars from CEG.
Location: The instrument is located in Rm 253 Kettering
Training mainly involves basic instrument and software manipulation to allow new users to start sample measurement independently. At least one-hour instrument demonstration and one-hour guided hands-on sample running are required prior to independent usage. Users can request more training hours if they are unable to fully perform the assays independently.
Trained users can reserve and access the instrument from 8:00 am to 5:00 pm, Monday through Friday. Usage may be available after working hours at a case-by-case scheduling base. Users are responsible for the correct manipulation of the instrument to achieve results and avoid damages.
Staff-operated assays A staff is available to run pre-prepared samples. This service is currently offered at restricted time. Currently, the service is open every week for two-three hours slots on Monday and Wednesday afternoons.
Consultation Users are encouraged to make appointments with Dr. Huang to discuss experimental design, pitfalls, and data analyses.analyses. Contact: Shouxiong Huang, PhD
Research supported by the Center for Environmental Genetics should acknowledge NIEHS Award P30 ES006096.
ImmunoSpot® Analyzers are designed for scanning and evaluating single cell-based spots, which reflect the protein expression, function, and growth of cells. Technically, the ImmunoSpot® S6 Micro’s zoom lens permits seamless switching from 6- to 384-well plates and counting objects <10 µm in size – including individual cells. With its maintenance-free 150-diode overhead lighting system and proprietary planar backlight system, the ImmunoSpot® S6 Micro provides the ultimate in image quality, especially for double-color assays (no fluorescence signal detected by this model). It is a low maintenance and high performance instrument for measuring functional responses and clustering growth derived from single cells at a high-throughput plate-based setting.
This ImmunoSpot® S6 Micro Analyzer can be applied to the following CEG-related or supported studies involving in environmentally induced diseases and conditions. (i) Characterization of cytokine, surface markers, or soluble molecules from rare cancer stem cells with high sensitivity and accuracy to capture individual cells. (ii) Functional measurement of the cytokine production from rare immune cell subsets, e.g. regulatory T cells, anti-inflammatory immune cells, etc, from human samples in response to toxicant-treatment or environmental exposure. (iii) Many reagents kits have been commercially developed for testing cytokine production that can be conveniently applied in toxicology studies. (iv) The growth of microbes from various pathogenic sources or from complex microbiota pools can be recorded, quantified, and differentiated using the high-resolution camera and analytical software, which can be applied for studying the environmental toxicity on microbiota. Notably, the ImmunoSpot® Analyzer is highly advantageous in functional characterization of rare cells from limited human samples and in the survey of rare subsets from exposure-related epidemiology studies using high-throughput microplate analyses.
Location: The instrument is located inRoom 253 Kettering Laboratory Building
Training mainly involves basic instrument and software manipulation to allow new users to start sample measurement independently. Users can access online resources from manufacturers or vendors for available reagents, general protocols, and overall manipulations. At least one-hour instrument demonstration and hands-on sample running are required prior to independent usage. Users can request more training hours if they are unable to fully perform the assays independently.
Trained users can reserve and access the instrument from 9:00 AM to 5:00 PM, Monday through Friday. Usage may be available after working hours at a case-by-case scheduling base. Users are responsible for the correct manipulation of the instrument to achieve results and avoid damages.
Consultation Users are encouraged to make appointments with Dr. Huang to discuss experimental design, pitfalls, and data analyses. Contact: Shouxiong Huang, PhDSign-up/reservation form
The Confocal Imaging service is equipped with “Carl Zeiss Confocal Laser Scanning Microscopy” LSM700.
Confocal Laser Scanning Microscopy combines high-resolution and high-contrast optical imaging with depth selectivity allowing visual sectioning of tiny structures and constructing three-dimensional (3D) structures from the obtained images. It is broadly used to resolve the detailed structure of specific objects within the cell; it also enables the creation of sharp images of the exact plan of focus without any disturbing fluorescent light from the background or other region of specimen. Stacking several images from different optical plans allows visualization and analyses of 3D structures. However, due to limited depth of sample penetration, the thickness of specimen is an important consideration for experimental success. Use of confocal microscopy in the biomedical sciences includes the imaging of multiple labeled specimens and the measurement of physiological events in living cells. Visualization of sufficient variants within both cell and tissue context will allow spatial and dynamic analyses of cell biology and may lead to advancement in understanding of biological processes.
Contact: Chia-I Ko, Ph.D. (email@example.com)
Location: Kettering laboratory complex, Room 443 (4th floor, Kettering Addition).
Instruction: Microscope use is free of charge only for CEG members. The sign-up/reservation form is mandatory for each use (link under development; in the interim, please contact Dr. Ko). Individuals who have received the microscope training are allowed and will be approved to schedule a reservation. New users should contact Dr. Ko to schedule an initial training before planning for experiments. Other services such as sample preparation instructions and troubleshooting for image-taking can be provided upon request; however, we do not guarantee for expected results. In case system failure is detected due to carelessness and mishandling, users will be charged for repair costs.
Versatile Imaging Systems
Odyssey imaging Systems: The Odyssey CLx is the next generation multifunctional imaging platform that can provide a wide range of applications. The Odyssey CLx platform uses infrared laser excitation that out-performs LED and visible white light systems for Western blots. This increased sensitivity offers a clear image of your data that is unmatched by other digital imaging systems. Two separate lasers and detectors simultaneously detect both fluorescent signals. The optical system employs diode lasers and solid-state detectors with long lifetimes and very low maintenance requirements. Infrared laser excitation outperforms systems that use white light, LED light sources, and filter wheels by delivering higher intensity excitation light to the fluorophore. A variety of fluorescent dyes and stains are compatible with the 700 nm and 800 nm excitation wavelengths of the two diode lasers in the Odyssey CLx. Spectral overlap is minimized by a 100 nm separation of the two detection channels, and optical filtering ensures that each detector measures fluorescence from only one of the infrared dyes
- See your bands reliably with stable fluorescent signals. Fluorescent signals are stable and unaffected by timing, so you can compare band intensities with confidence. You can even conveniently re-image the same blot on the Odyssey CLx later and see the same results.
- See multiple targets on the same blot. With multiplex fluorescence, you can detect two protein targets in each sample lane, with great sensitivity in both fluorescence channels.
- See your strong bands accurately and immediately. Get deeper data capacity with digital fluorescence and the unprecedented linear dynamic range of the Odyssey CLx, and never lose data because of image saturation.
- Get better sensitivity with low image background. Powerful, precise laser excitation and specialized optics enable the Odyssey CLx to provide high signal-to-noise ratios and outstanding image quality.
- See the full story in a single image. See both strong and faint bands clearly in a single image acquired on the Odyssey CLx, with great sensitivity and no image saturation. Multiplex with two fluorescent colors to see even more data.
- Do more with digital fluorescence. Get consistent, accurate digital images, without the hassles and unpredictability of film – so you can analyze your data right away and plan your next experiment.
- Stable NIR fluorescence and detection of multiple targets in the same lane. Get the most accurate results with over 6 logs of linear dynamic range, so you can make more discoveries.
- In-cell western capability present. However, user will have to develop protocol and initiate use of this facility.
Training: Training will mainly involve basic instrument and software manipulation to allow new users to start taking western-blot images independently. At least 10 min instrument demonstration and first time guided hands-on software running are required prior to independent usage. For Odyssey-CLx knowledge, experimental design, data interpretation, and result presentation, users should refer to tutorials available on request. Also refer to https://www.licor.com/bio/webinars/ , https://www.licor.com/bio/odyssey-clx/resources before starting experiment.
Scheduling: After being trained and able to perform assays independently, users can contact our staff for scheduling. Usage slots of more than one hour must be justified. Users are also required to sign on the usage page and provide project information for CEG.
Sample format: Western blot membranes and reagents should be provided by users. First time users of this NIR western blotting technique can request use of secondary antibodies and blocking solutions from Dr. Tarapore.
Fees: Available to CEG members without charge
Data Storage: A project drive, called “CEG_Users”, has been set up for temporary storage. If the computer space is limited, the stored data will be removed after 6-month from initial acquisition.
Fluidigm Automated Systems
C1 isolates single cells into individual reaction chambers in the exclusive Fluidigm integrated fluidic circuit (IFC). The IFC automatically stain captured cells and examine them by microscopy for viability, surface markers or reporter genes. After staining, cells are automatically lysed and template is quickly prepared for qPCR or sequencing library preparation for mRNA sequencing, DNA sequencing, epigenetics or miRNA expression.
JUNO, Automated NGS library prepartion via Fluidigm JUNO system for RNA-seq and ATAC-seq.
BIOMARK, TaqMan-based qPCR via Fluidigm Biomark HD system for analyses such as gene and miRNA exression.
Brochure (BIOMARK HD)
Contact: firstname.lastname@example.org, Kettering room 336
Instructions: We follow the general instructions used by others. Users should be advised that the system is complicated. Potential risks of the system or experiment failure exists. The system does not have a service contract.