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Axle Informatics, LLC is currently seeking a Scientific Operator for Flow Cytometry Instruments for the National Institute of Health (NIH); Bethesda, Md.
Overview
Scientific Operator for Flow Cytometry Instruments
Purpose
To provide an outstanding scientific operator for flow cytometry instruments at the National Institutes of Health in Bethesda, MD. The Flow Cytometry Facility in the Research Technologies Branch (RTB), in the Division of Intramural Research (DIR), National Institutes of Health. The research agenda of the DIR focuses mostly on immunology and infectious disease research, and flow cytometry is central to many research projects. The DIR supports approximately 120 principle investigators and 500 postdoctoral and clinical fellows. The Facility currently hosts six high speed cell sorters, five cell analyzers, and one imaging cytometer. Operations covers both BSL2 and BSL3 laboratories.
Responsibilities and Duties
- Contribute to all aspects of facility operation, including flow cytometry analysis and cell sorting with added emphasis on independent operation of cell sorters inside both BSL2 and BSL-3 labs.
Qualifications
- Positions require bachelor’s or master’s degree in Biology, Immunology, or related field.
Development of new methods that extend the scope of flow cytometry at the facility will require excellent laboratory skills and a high level of desire to learn new skills
For additional questions or concerns please contact Stefonie Kelly at stefonie.kelly@axleinfo.com or 202.421.2402.
greg.inguagiato@resourcesolutions.com
Senior Scientist – Flow Cytometry
Valencia, CA
R1038817
Job Summary
Resource Solutions is working on behalf of our client, Q2 Solutions, the world’s second largest
clinical laboratory services provider. Launched in 2015 through a joint
venture between Quintiles and Quest
Diagnostics, Q2 Solutions is focused on helping
biopharmaceutical, medical device and diagnostics customers improve human health
through innovation that transforms science and data into actionable medical
insights.
Responsibilities
- Assist with optimizing and improving flow cytometry
operations
- Perform routine technical troubleshooting of flow
cytometry instruments and assays
- Perform bench work and routine gating for operational
support
- Performs data analysis and reporting of flow cytometry
clinical samples across a variety of cytometry assays.
- Provides technical expertise, leadership and support
for flow cytometry operations for assay/result troubleshooting.
- Reviews and documents the results of technology
transfers and process improvement validations. Collaborates with the
Translational Sciences Laboratory where appropriate.
- Investigate and resolve operational and quality issues
within the flow cytometry department.
- Write and review standard operating procedures and
policies for the department.
- Assist in the qualification and validation of new or
repaired instruments.
- Assists with training for flow cytometry staff for new
and existing assays.
- All responsibilities are essential job functions unless
noted as nonessential (N).
Required Knowledge, Skills and Abilities
- A good understanding of equipment, software and
processes of flow cytometry and the overall subject area.
- Knowledge of clinical and/or research laboratory
environment and quality control procedures.
- Excellent Excel skills.
- Ability to balance multiple priorities and work to
short deadlines.
- Ability to establish and maintain effective working
relationships with co-workers, managers and clients.
- Good oral communication skills.
Minimum Required Education and Experience
- Bachelors degree in chemical, physical, biological
science, or clinical laboratory science
- 5+ years of laboratory related experience with Flow
Cytometry, experience in regulated clinical lab is preferred
- CLIA and ASCP certification is highly preferred, not
required
About our Client
With more than 9 million
clinical laboratory tests managed annually, we are committed to providing our customers an innovative,
progressive and responsive partner with the quality focus, global experience
and deep medical expertise integral to drug, medical device and diagnostic
development.
We work collaboratively with our customers,
business partners and colleagues to lead the industry and live our customer
promise of providing Actionable
Insights for Better Health™.
Our commitment to innovation is how we’re
building better clinical laboratory services for our customers’ – we call it
“the lab of the future.” The lab of the future will help our customers develop
drugs faster and more efficiently. Learn more about how we partner with our
customers to build the lab of the future through: Tailored Solutions, Delivery
Excellence and Shaping Outcomes.
What we Offer
·
Major Medical, Vision, Dental, Disability
·
Annual bonus
·
401(k) with 100% vesting after 2 years
·
Tuition Reimbursement up to $6k annually
·
Generous paid time off and holiday leave
·
Other generous benefits
EEO Minorities/Females/Protected Veterans/Disabled
Does anyone know if there is a Euroflow users group?
We are experiencing some issues with EQA material on a certain EQA program. The same issues are not present in our patient material.
What EQA programs do other people use - especially on Euroflow?
Any body having issues?
Almero
South Africa
We have available a unique cell-tracing product to the pharmaceutical industry that enables 10 × higher visibility and dramatically longer tracing of cells than any other product on the market today. If interested please respond.
Silicon Microchip Capable of High-Frequency Fluidic Valving at Heart of Technology
Jim Linton, Ph.D. Shane W. Oram, Ph.D.
The purification of cellular populations and individual cells is pivotal for the reliable characterization of gene expression, many aspects of life science research, and the development of cellular therapies. Cell sorting routinely involves fluorescence-based separation through flow cytometry, which has proven superior to crude techniques such as differential sedimentation. Through this process, large numbers of cells are rapidly analyzed for specific fluorescence signatures. Traditionally, cell samples are parsed in charged aerosol droplets, which are electrostatically sorted, enabling purification at thousands of cells per second at purities often greater than 90%.
This technology enables exceptional specificity using multiple fluorescent signatures (e.g., cell surface labels, cell size, and granularity).
However, the contributions of current flow-sorting platforms are balanced against significant limitations, including: high processing pressures that can result in loss of function and/or cell death; sample processing speeds/volumes that make processing clinical-scale samples (>500 million cells) unfeasible; a high degree of technical expertise needed to manage device complexity; increased risk of sample contamination through the use of open systems; and user safety concerns when processing aerosolized patient samples.
These limitations, plus the high unit and sample processing costs, must be overcome to enable further clinical application and commercialization.
To address these issues, Owl Biotech developed a fully closed cell-sorting system. This microchip-based technology employs closed fluid path cartridges with aseptic ports that permit the straightforward administration and collection of cell samples. At the heart of the cartridge is a patented microchip capable of very high-frequency fluidic valving (Figure 1).
Propelled by modest positive pressure, typically less than 0.2 atmospheres, cells pass through microchannels where laser-directed fluorescent signals are detected with photo-multiplier tubes. Upon identification of a positive target cell, the microchip valve opens, redirecting the cell to a collection chamber. Both positive and negative selected cells can then be retrieved from the cartridge and used for any number of downstream applications.
Improving Valve Speed Click Image To Enlarge +
Figure 2. Mechanism of microchip-based sorting: Labeled cell samples enter the chip from the input sample, as the cells approach the sort area each cell is analyzed. When a selected cell is identified a magnetic pulse opens and closes the valve and the cell is redirected to a collection chamber. An integrated single-crystal silicon spring returns the valve to its original position, and undesired cells are allowed to flow through.
One key design goal of all cell sorters is to maximize the speed at which the device can segment a stream of cells. In the case of Owl’s microchip-based technology, a fluidic valve determines the rate at which cells are isolated. Valve speed in a fluid microenvironment is in turn controlled by several factors, including the acceleration and magnitude of the opening and closing forces, and the inertia of the valve and the fluid surrounding it.
In the case of Owl technology, valve speed is controlled by its engineered magnetic properties and a powerful return-spring force, which serves to close the valve (Figure 2). Careful modeling and empirical testing has led to a design that allows μsec opening times, a user-selected sort collection delay, and μsec closing times.
A typical total cycle of around 50 μsec allows separation rates similar to a traditional droplet sorter, although with microchip-based sorting no aerosols or droplets are used.
Sorted cells are typically used for molecular analysis or sample preparation, for example, cellular expansion for research or therapeutic purposes. In such cases, those cells need to be in a healthy metabolic state ideally retaining their complete array of functional capabilities. Current flow cytometric sorting has distinct challenges in that respect due to technical requirements such as high pressure, extended shear rate, severe decompression upon aerosolization, and impact trauma during cell collection. Often, these factors result in cell isolates with compromised function and/or viability.
Using Owl’s microchip-based technology, the pressure applied to cells is minimal and the trauma associated with droplet sorting is removed, resulting in high cell viability. In addition, a wide variety of cell types have been sorted using the Owl technology, all with a high retention of cell functionality. For example, antigen-primed T cells have been shown to retain their cancer-specific cytotoxic capabilities in chromium-release assays.
Figure 3. Sorting target cells from dilute whole blood or PBMCs: Pre-sort fraction of diluted whole blood with CD4+ cells (A) Post-sort fraction showing enrichment of CD-4 positive cells (B) Pre-sort fraction of the spiked MART-1-specific T-cell clone into PBMCs (C) Post-sort fraction showing enrichment of MART-1 positive antigen specific T cells (D).
While the utility of microchip-based cell separation is being tested in many different applications, current studies show its utility in processing clinical samples for diagnostic and therapeutic applications. For these purposes it is best to process the samples with as little manipulation as possible.
Here, CD4+ cells were sorted from a diluted whole blood preparation by adding a fluorescent CD4 antibody marker to 8 mL PBS and 2 mL of whole blood. Results show that while the presorted fraction contained less than 0.01% CD4 positive cells, the sorted fraction contained more than 91% CD4 positive cells (Figures 3A and B). The effective purification was close to 10,000-fold in a single-step process and with a simple no-lyse, no-wash sample preparation.
Investigations have also been done to study the ability of antigen-specific T cells from a patient’s own blood to recognize tumor cells and trigger an immune system response. To explore the feasibility of this clinically important strategy, cells from a MART-1-specific T cell clone were spiked into a patient’s peripheral blood mononuclear cells (PBMCs), stained with a PE-conjugated tetramer loaded with a MART-1 peptide, and then sorted using the Owl’s microchip-based sorting technology.
Results show tetramer-positive cells were enriched from less than 1% to greater than 95% (Figures 3C and D). Importantly, sorted cells maintained their ability to kill MART-1+ tumor cells and to proliferate in vitro.
Acceleration of research for effective cancer and cellular therapies has increased the need for cell-sorting technologies that are safe, efficient, and easy to use. Owl’s cartridge-based closed system has effectively demonstrated its capability to sort a wide variety of cells at high speeds without impacting cell viability. This platform offers the additional advantage of protecting sample integrity while permitting quick sample-to-sample changeover—a feature that avoids the inter-sample cleaning and validation required for traditional cell sorting.
That this technology does not utilize sheath fluids opens the future potential for sequential sorting of large numbers of samples within a short timeframe, making practical clinical applications a possibility. In addition, combining the underlying microchip technology with other cell isolation techniques such as magnetic beads has the potential to empower processing of samples greater than 1 billion cells.
Jim Linton, Ph.D. (jim@owlbiomedical.com), is chief business officer at Owl biomedical, and Shane W. Oram, Ph.D. (shaneo@miltenyibiotec.com), is global marketing manager—cell analysis at Miltenyi Biotech.
Source: https://technical.sanguinebio.com/current-options-for-isolating-pure-cell-populations/
Author: Colt Egelston
Antibody based isolation kits for isolating immune cell populations have become a standard protocol in the toolbox of every immunologist over the last two decades. In fact, many new scientists are shocked to learn that lymphocytes used to be isolated from PBMCs and other tissue sources by filtering through nylon wool. How archaic! Here I will describe the various options cell isolation technologies available to biologists today.
FACS: Fluorescence Activated Cell Sorting
FACS is the most sophisticated way of isolating various cells of interest from your tissue source. You have the ability to incorporate up to 10 or so different fluorescent antibodies into your stain, which allows you to sort on cells of interest with exquisite precision and specificity. Another powerful tool is the ability of many FACS machines to do four-way sorts or even single-cell sorts.
However, sorting can be relatively time consuming, depending on your sample size and the percentage of cells of interest. Use of FACS machines are also fairly expensive, whether it be your laboratory’s investment in acquiring its own machine and committing to its maintenance or the hourly rates your institution’s core will charge you (averaging around $100 per hour in my experience).
Magnetic Antibody Based Cell Isolation
Cell separation reagents are available from the three main players in the cell isolation kit world: Stem Cell Technologies, Miltenyi Biotec MACS Technology, and Life Technologies Dynabeads. Though the technology varies slightly from company to company, they basically boil down to the same principles. Usually an antibody cocktail will bind either your cell of interest (positive selection) or your cells of non-interest (negative selection). After a short incubation the addition of magnetic nanoparticle beads to your cell mixture then binds the antibodies from the previous incubation. After another short incubation, cells can then be placed into the magnet purchased from the company. After a few minutes, the antibody bound cells will be drawn towards the magnet and the unbound cells can be collected. Bound cells can then be washed out and collected separately. This technology allows rapid and easy isolation of cell populations from bulk populations.
However, magnetic antibody based cell isolation involves some upfront investment in the purchasing of magnets (approaching $1000) and antibody kits (ranging from $300-$700). Because of this it is important to fully research which companies’ technology is right for you. I also highly recommend sampling the technology on some extra PBMCs you have if at all possible and finding an experienced colleague that can advise when you have questions.
RosetteSep Whole Blood Based Cell Isolation
RosetteSep kits from Stem Cell Technologies allow researchers to quickly isolate cells of interest directly from whole blood and without the investment in magnets. Furthermore it combines the Ficoll gradient isolation step with the isolation of specific target cells, making for an efficient and economical protocol. Instead of using magnetic nanoparticles, RosetteSep uses antibodies that conjugate directly to the RBCs in whole blood. When the blood is Ficolled the RBCs go to the bottom layer along with all the cells that you have targeted with antibody. Your top layer is left with untouched cells of your interest! Of course this protocol only works from whole blood, so it will not work on PBMCs or cells from other tissue sources.
Keep in mind that both FACS and antibody based cell isolation require starting with a single cell suspension of cells. It is important to think about whether you want touched or untouched cells (positive or negative selection) for your downstream assays. I also highly recommend doing purity checks (see figure below) by flow cytometry as often as you can, especially when first adapting any isolation technology to your lab.
Further Reading:
Stem Cell Technologies: https://www.stemcell.com/en/Products/Product-Type/Cell-isolation-products.aspx
Life Technologies Dynabeads: https://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Dynal/Dynabeads-Types-and-Uses.html
Miltenyi Biotec MACS Technology: https://www.miltenyibiotec.com/en/Products-and-Services/MACS-Cell-Separation.aspx
RosetteSep: https://www.stemcell.com/en/Products/Popular-Product-Lines/RosetteSep.aspx
About the Author:
Colt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.
