| Flow Cytometry |
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The Flow Cytometry Unit provide a range of services, including sample analysis and cell sorting, as well as equipment, software, and technical expertise/advice for a variety of flow cytometric protocols.
Its function is to provide high quality, cost effective state-of-the-art flow cytometry and multiparameter cell sorting to all investigators at CNC and serves to enhance the quality and scope of scientific research performed at CNC.
The unit has a state of the art cell sorter FACSAria III, where as much as four different cell populations can be sorted simultaneously, and cells can either be sorted onto slides, into tubes (1-15 ml) or plates (6-384 wells). In addition, we have a Partec CyFlow space that sort one population at a time, and a FACSCalibur and an Accuri C6 analyser, which can perform multi-parameter flow cytometric analysis.
Training is available to enable users to run analytical cytometers themselves. The flow cytometry unit is available to all CNC researchers, as well as to other interested academic or commercial users, after prior arrangement.
Guidelines for the use of the facility are provided on these web pages. Please ensure you have read the relevant guidelines to maximize the success and validity of your experiment.
Technologies/Services:
 Cell sorting;
Data acquisition, analysis and interpretation;
User training;
Advice on sample preparation;
User assistance and machine troubleshooting;
Help with design of experiments;
Sourcing of reagents and techniques and finding alternative approaches;
Help with data preparation for presentation and publication;
We are also open to collaboration.
List of equipment available:
Becton Dickinson FACSCalibur cell analyser (Polo I);
Partec CyFlow® Space cell sorter (Polo I);
Becton Dickinson Accuri™ C6 cell analyser (UC-Biotech) with auto-sampler;
Becton Dickinson FACSAria III cell sorter (UC-Biotech).
Flow Cytometry Unit Access Rules
The responsible technician must attend a new user first flow cytometry session.
Cell Analysers are available 7 days a week-24 hours a day for trained users. Cell Sorters require a dedicated operator and are open for use during normal Monday to Friday working hours.
Before using the flow cytometers, the user must have a valid reservation in the booking system.
If you find a problem with the flow cytometer or evidence of incorrect usage immediately contact the responsible technician.
Booking
New users should send an email to flow-poloI@cnc.uc.pt (Polo I) or flow@uc-biotech.pt (UC-Biotech), to schedule the first flow cytometry session and training.
To book go to the Agendo-Center for Neuroscience and Cell Biology web page: https://cnc.agendo.science and follow the instructions (Polo I) or contact flow@uc-biotech.pt (UC-Biotech).
Users must always use their own reserved time and should not use another user account to book time. Use without a reservation is prohibited.
“No-shows” are charged for all of the scheduled time.
Sorting or Analyzing Potentially Biohazardous Samples
The facility cannot accept potentially pathogenic samples and prior approval for sorting or analyzing viable human/primate samples, cells infected with replication incompetent virus, lentivirus-treated samples, or cells from animals previously exposed to an infectious agent, should be obtained from the responsible of the facility. Samples labeled with radioactive materials are not accepted under any circumstances.
Publication Acknowledgments
To obtain funding and sponsorship, we must be able to demonstrate that the Unit provides a useful service and enhances research at CNC.
In order to help us recognize the value of the Facility to your research, please acknowledge use of the Unit in all publications.
A suggested text is: “We acknowledge the assistance of the CNC Flow Cytometry Unit, where flow cytometry experiments were performed.” In the event that Facility staff has contributed significantly to specifics of experiment design, data analysis or provided specialist technical troubleshooting beyond the norm, please mention this in the acknowledgements. Please notify the platform coordinator of any publications, and email the reference details.
Investigator Responsibilities for Running Samples
Cells used to set up the instrument must be the same or very similar to the experimental samples. For example, you cannot use resting lymphocytes to set up the instrument and then run a cell line for analysis. The auto fluorescent properties of the cells types are too dissimilar. Setting up the instrument consists of running a negative control and compensation tubes so that amplifier gains can be properly set for subsequent samples.
The cell preparation must be as clean as possible regarding debris and aggregates. Large amounts of debris and/or aggregates can obscure the population of interest, making forward scatter and side scatter amplifier gain settings difficult to set correctly.
The investigator is responsible for having the correct cell density (1 × 106 per ml) and volume (minimum of 300 ul) per tube for analysis. Cell density for sorting should be between 5 × 106 and 20 × 106 per mL.
The person accompanying the tubes should have intimate knowledge of the samples and what the data may look like when correctly run.
Compensation Controls for Flow Cytometry
Flow Cytometry technology can yield highly accurate data, but only if the proper controls are done prior to sample analysis. This is especially important when multi-color analysis is undertaken. Spectral overlap between fluorochromes in multi-color experiments requires the use of compensation (see http://www.drmr.com/compensation/index.html for an in depth explanation of compensation). Without proper compensation, the data obtained would be meaningless. The proper compensation controls may vary from experiment to experiment, but some basics are the same. Thus, an unstained or isotype control and a positive control for each color are essential.
Experiments that cannot spare cells for compensation controls can use a bead product from Becton Dickinson called BD CompBeads. If the beads are used, the experiment would only require the addition of an unstained or a negative tube of cells to set the initial baselines.
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