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It’s HOW you count your cells that matter

 :: Posted by American Biotechnologist on 10-15-2010

For over 100 years the hemocytometer has been used by cell biologists to quantitate cells. It was first developed for the quantitation of blood cells but became a popular and effective tool for counting a variety of cell types, particles and even small organisms. Currently, hemocytometers, armed with improved Neubauer grids, are a mainstay of cell biology labs.

Despite its longevity and versatility, hemocytometer counting suffers from a variety of shortcomings. These shortcomings include, but are not limited to, a lack of statistical robustness at low sample concentration, poor counts due to device misuse, and subjectivity of counts among users, in addition to a time-consuming and tedious operation. In recent years automated cell counting has become an attractive alternative to manual hemocytometer-based cell counting, offering more reliable results in a fraction of the time needed for manual counting.

The attached report compares the precision of cell counts obtained with a hemocytometer to hose obtained by automated cell counting using the Bio-Rad TC10 automated cell counter. Sources of error that are inherent to the device, and those introduced by the operator are investigated. We demonstrate that automated cell counting can significantly reduce user and concentration-dependent count variance, while greatly reducing the time needed to perform counts.

Comparison of a Hemocytometer and TC10 Automated Cell Counter

Multiplexing Bio-Plex Pro Diabetes and Cytokine Assays

 :: Posted by American Biotechnologist on 07-01-2010

In a previous post we introduced Bio-Rad’s Bio-Plex Pro Diabetes and Cytokine assays and mentioned how one of the biggest advantages that the kit offers to researchers is the ability to multiplex cytokine and diabetes biomarkers in one tube. In the attached tech note: Multiplexing Compatibility of the Bio-Plex Pro Diabetes and Cytokine Assays: Human and Mouse Panels, Zimmerman et al. demonstrate the compatibility of 40 human cytokine assays with all 10 human diabetes assays and 32 mouse cytokine assays with all 8 mouse diabetes assays. The data includes an analysis of sensitivity, specificity and accuracy with limits of detection as low as 0.1pg/ml for several cytokine and diabetes biomarkers.

Multiplexing Compatibility of the Bio-Plex Pro Diabetes and Cytokine Assays: Human and Mouse Panels

Simplifying Gene Knockdown Experiments

 :: Posted by American Biotechnologist on 05-17-2010

If you’ve ever done gene knockdown experiments, you know that the process can be complicated and involve many steps. siRNA design, cell culture, perfect cell confluency, transfection or electroporation, downstream analysis etc. Bio-Rad now has a plethora of instruments and reagents to help you simplify the process. In a recently published article in the Journal of Visualized Experiments, scientists demonstrated a pathway study that makes use of Bio-Rad’s many strengths in the area of cell culture and gene silencing including streamlining cell counting with the TC10 automated cell counter, electroporating multiple samples simultaneously using the MXCell electroporation system and simultaneously assessing RNA quality and quantity with the Experion automated electrophoresis system.

If you want to learn about more ways to simplify your siRNA experiments checkout:

McCoy, A., Litterst, C., Collins, M., & Ugozzoli, L. (2010). Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells Journal of Visualized Experiments (38) DOI: 10.3791/1904

How to Enrich Low Abundance Proteins from Tissues, Cell Lines and Bacteria

 :: Posted by American Biotechnologist on 05-06-2010

Biotechnologists engaged in protein biomarker studies are well aware of some of the challenges they face trying to identify biomarkers in albumin and IgG rich serum and plasma. Protein biomarkers generally tend to be low-abundance proteins which are very difficult to detect among highly complex samples. Furthermore, an ever-increasing amount of biomarker studies are focused on identifying biomarkers in other biological sources such as muscle tissue and cell lines. Traditional approaches to solving this problem include depletion methods for removing high abundant proteins so that the proverbial “needle in the haystack” biomarker is easier to locate. While this approach has its advantages, it suffers from being too specific and may not work well in sample types other than serum and plasma.

Bio-Rad Laboratories Inc. has developed the ProteoMiner protein enrichment technology which uses a library of hexapeptides to bind all proteins in a complex mixture allowing reduction of high-abundance proteins and enrichment of medium and low-abundance proteins.

This Tech Note discusses the use of ProteoMner protein enrichment kits for the enrichment of low- and medium- abundance proteins and the depletion of high-abundance proteins in tissue, cell line and bacterial samples, with detailed experimental parameters in a 2-D gel based proteomics workflow.

Enrichment of Medium- and Low-abundance Proteins

How to Perform Highly Efficient Reverse siRNA Transfection

 :: Posted by American Biotechnologist on 05-03-2010

Short interfering RNAs (siRNAs) are powerful tools to suppress gene expression in mamalian cells. While siRNA transfection can often be bogged down by cell seeding and density the highlited study demonstrates a highly efficient reverse siRNA transfection protocol for A549 cells using Bio-Rad’s siLentFect lipid reagent that does not require prior seeding of cells. Furtheromore, the simplicity of the protocol makes it suitable for high-throughput transfection. The authors acheived an average of 94% knockdown for the two genes that were targetted without impairing cell viability.

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