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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.

Perform daily Quality Control and maintenance.

Perform troubleshooting of Flow Cytometry equipment.

Provide support to researchers in operating analyzers.

Assist with user training and basic panel development.

Perform sample preparation and staining for flow cytometry analysis.

Assist in the preparation of SOPs and related documents for GLP work.

Comply with safety guidelines and requirements by following and ensuring strict safety procedures and safety checks.

Assist in Research and Development programs for new assays and technologies 

Qualifications

• Positions require bachelor’s or master’s degree in Biology, Immunology, or related field.

Minimum five (5) years or more of demonstrated relevant experience in flow cytometry.

Experience in the operation of cell sorters is highly preferable.

Proficiency with basic laboratory techniques (sample preparation, cell culture) and flow cytometry methods such as immunophenotyping, cell cycle analysis, etc.

Experience in high parameter flow cytometry will be an advantage, but training can be provided where required.

Good understanding of laboratory biosafety standards.

Proficiency in the use of Microsoft Office including knowledge of computer systems and ability to work independently with data analysis programs

Ability to multi-task in a busy facility and to approach tasks and obstacles with a team-first mentality.

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.

if you have a passion for Flow Cytometry and want to join a progressive company, give me a call. We are adding new sponsors and increasing our service offerings to existing sponsors.   Full time roles with benefits. Greg Inguagiato 904-831-5682

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

 

 

 

Dear All

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 are going to distinguish NKcell population within PBMC through flow cytometry. Our approach is as follows:- 1. From the dot plot of FSC vs SSC we r gating CD-3 Cy5|| CD-56 FITC. 2. To isolate NK cell We are choosing the CD3-/CD56+ cells. 3. As we wish to detect the NK cell dim/bright population, we then gate CD56 FITC||CD16PE population. 4.At this stage we have isolated NK population and also differentiated NK dim/bright cells also. 5. Next we are going to observe CD-328 PE along with CD56 FITC dim/bright. 6. Similarly CD-329 PE will be observed. IMPORTANT NOTE: Our constraints:- 1.we can not perform 6 color facs. 2. We have isotype control having 3 fluorophores namely FITC, PE, Cy5 which can't be altered due to budget constraint. 3.we have access to a BDFacscalibur having 4color only namely FL1,FL2,FL3,FL4. We prefer to perform 2color face. 4. Due to budget constraint , we will set the CD3 and CD16 gates first and then further just gate the PBMC through the previously selected gate. Then we will just tag the PBMC with CD56 & CD328/CD329 is it okay? Waiting for your kind reply.

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.

Source: https://www.genengnews.com/gen-articles/next-generation-flow-sorting/4904/

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.

Highly Viable Sorted Cells

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.

Microchip-Based Sorting Click Image To Enlarge +
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.

Conclusions

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.

These powerful techniques allow for biologists to isolate a host of cells, including T cells, B cells,  Monocytes, Stem Cells, and many more. In an upcoming post I will go into even further detail and how to choose the right technology for you, including some of the tips and tricks I have learned in my own experience

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.

Source: https://www.bioopticsworld.com
08/27/2013

Posted by Lee Mather

Associate Editor, BioOptics World

Scientists at McGill University (Montreal, QC, Canada) have crystallized a short RNA sequence, poly (rA)11, and used data collected at the Canadian Light Source (CLS; Saskatoon, SK, Canada) and the medical diagnostics (Ithaca, NY) to confirm the hypothesis of a poly (rA) double helix. Their discovery, building on 50 years' worth of work by various scientists, will have interesting applications for research in biological nanomaterials as well as in fabricating bionanomachines (devices derived from living organisms that can perform Related: AFM collaboration produces first in-situ view of DNA's double helix).

https://onlinelibrary.wiley.com/doi/10.1002/anie.201303461/abstract

“Bionanomachines are advantageous because of their extremely small size, low production cost, and the ease of modification,” explains Kalle Gehring, a biochemistry professor at McGill University who led the work. “Many bionanomachines already affect our everyday lives as enzymes, sensors, biomaterials, and medical therapeutics.”

Gehring adds that proof of the RNA double helix may have diverse downstream benefits for the medical treatments and cures for diseases like AIDS, or even to help regenerate biological tissues. His team initially was looking for information about how cells turn mRNA into protein when they made their discovery.

For the experiments, Gehring and a team of researchers used data obtained at the CLS Canadian Macromolecular Crystallography Facility (CMCF) to successfully solve the structure of poly (rA)11 RNA. CMCF Beamline Scientist Michel Fodje said the experiments were successful in identifying the structure of the RNA and may have consequences for how genetic information is stored in cells.

Structure of poly (rA) duplex showing the two strands in orange/yellow and green/blue. Ammonium ions that stabilize the structure are shown as black balls. (Image courtesy of Kathryn Janzen, Canadian Light Source) “Although DNA and RNA both carry genetic information, there are quite a few differences between them,” says Fodje. “mRNA molecules have poly (rA) tails, which are chemically identical to the molecules in the crystal. The poly (rA) structure may be physiologically important, especially under conditions where there is a high local concentration of mRNA. This can happen where cells are stressed and mRNA becomes concentrated in granules within cells.”

With this information, researchers will continue to map the diverse structures of RNA and their roles in the design of novel bionanomachines and in cells during times of stress.

Research on the poly (rA) structure was funded by grants from the Natural Sciences and Engineering Research Council of Canada with support from the Canada Foundation for Innovation, the Government of Quebec, Concordia University, and McGill University.

The research team's paper appears in the journal Angewandte Chemie International Edition; for more information, please visit https://onlinelibrary.wiley.com/doi/10.1002/anie.201303461/abstract.

(Source: https://www.sciencedaily.com/releases/2013/07/130710141854.htm)

There are several ways to "trap" a beam of light -- usually with mirrors, other reflective surfaces, or high-tech materials such as photonic crystals. But now researchers at MIT have discovered a new method to trap light that could find a wide variety of applications.

The new system, devised through computer modeling and then demonstrated experimentally, pits light waves against light waves: It sets up two waves that have the same wavelength, but exactly opposite phases -- where one wave has a peak, the other has a trough -- so that the waves cancel each other out. Meanwhile, light of other wavelengths (or colors) can pass through freely.

The researchers say that this phenomenon could apply to any type of wave: sound waves, radio waves, electrons (whose behavior can be described by wave equations), and even waves in water.

The discovery is reported this week in the journal Nature by professors of physics Marin Soljačić and John Joannopoulos, associate professor of applied mathematics Steven Johnson, and graduate students Chia Wei Hsu, Bo Zhen, Jeongwon Lee and Song-Liang Chua.

"For many optical devices you want to build," Soljačić says -- including lasers, solar cells and fiber optics -- "you need a way to confine light." This has most often been accomplished using mirrors of various kinds, including both traditional mirrors and more advanced dielectric mirrors, as well as exotic photonic crystals and devices that rely on a phenomenon called Anderson localization. In all of these cases, light's passage is blocked: In physics terminology, there are no "permitted" states for the light to continue on its path, so it is forced into a reflection.

In the new system, however, that is not the case. Instead, light of a particular wavelength is blocked by destructive interference from other waves that are precisely out of phase. "It's a very different way of confining light," Soljačić says.

While there may ultimately be practical applications, at this point the team is focused on its discovery of a new, unexpected phenomenon. "New physical phenomena often enable new applications," Hsu says. Possible applications, he suggests, could include large-area lasers and chemical or biological sensors.

The researchers first saw the possibility of this phenomenon through numerical simulations; the prediction was then verified experimentally.

In mathematical terms, the new phenomenon -- where one frequency of light is trapped while other nearby frequencies are not -- is an example of an "embedded eigenvalue." This had been described as a theoretical possibility by the mathematician and computational pioneer John von Neumann in 1929. While physicists have since been interested in the possibility of such an effect, nobody had previously seen this phenomenon in practice, except for special cases involving symmetry.

This work is "very significant, because it represents a new kind of mirror which, in principle, has perfect reflectivity," says A. Douglas Stone, a professor of physics at Yale University who was not involved in this research. The finding, he says, "is surprising because it was believed that photonic crystal surfaces still obeyed the usual laws of refraction and reflection," but in this case they do not.

Stone adds, "This is in fact a realization of the famous 'bound state in the continuum' proposed by von Neumann and [theoretical physicist and mathematician Eugene] Wigner at the dawn of quantum theory, but in a practical, realizable form. The potential applications the authors mention, to high-power single-mode lasers and to large-area chemical [and] biological sensing, are very intriguing and exciting if they pan out."