Cre-loxP Recombination Essentials Part 2

Cre-loxP Recombination Essentials Part 2

The Cre-loxP recombination system is routinely used for the generation of mouse knockouts. In part 1 of this mini-series, I introduced the concept and applications of Cre-loxP. As with any other technology or research tool, it has limitations and pitfalls that need to be considered while planning experiments or interpreting results. This article will take…

Cre-loxP Recombination Essentials Part 1

Cre-loxP Recombination Essentials Part 1

You might have heard of the Cre-loxP system even if you are not directly working with genetic manipulation. The Cre-loxP system is an ubiquitous technology for genetic manipulation and a mainstay in mouse research labs. With this system you can delete genes in cells, specific tissues and even whole organisms! You can start to master this system by…

Mini Me: What Makes for Good Models of Human Disease?

Mini Me: What Makes for Good Models of Human Disease?

One of the major roadblocks to the development of novel therapies is the lack of robust and reliable animal models. Selecting and validating animal models that mimic human conditions is challenging, especially when faced with chronic multi-factorial diseases such as diabetes and obesity. Acknowledging this problem, the National Institutes of Health initiated the Animal Models…

Simple Tips for Model Organism-Based Work

Simple Tips for Model Organism-Based Work The mouse is the favored model organism for life science researchers so much so that mice account for about 95% of all lab animals used in research. The striking similarities between the human and mouse genomes, ease of genetic manipulation and the uniformity achieved through inbred mating makes them…

Setting up a Fermentation or Perpetuum Mobile Cell Culture

Setting up a Fermentation or Perpetuum Mobile Cell Culture

Some names are confusing. For example, ant-lion is not an ant – or a lion. Likewise, fermentation in the scientific sense does not involve using a ferment or brewing beer. In science, fermentation is the setting up of a long-term culture of eukaryotic or prokaryotic cells. Fermentation is invaluable in providing a steady flow of…

How to perform cell synchronization in specific cell cycle phases

How to perform cell synchronization in specific cell cycle phases

The cell cycle has been very well documented over the years because of its dysregulation in diseases such as cancer. Many different processes contribute to cell growth and replication, which is ultimately controlled by a series of tightly controlled cell cycle phases. For some areas of research, especially within drug discovery and cancer research, cell synchronization in…

Laboratory Animal Ethics: The Three Rs

Laboratory Animal Ethics: The Three Rs

Animal models have helped make enormous discoveries and breakthroughs in the last few decades. From Pasteur’s use of sheep to test the ‘Germ Theory’, Pavlov’s classic conditioning experiments in dogs, to Dolly the first cloned mammal, animal research has come a long way. Today, most drugs, vaccines and other pharmaceutical products for medical use are…

Basic Bacterial Culturing Practices

Basic Bacterial Culturing Practices

Mastering basic bacterial culturing practices is a must if you are planning a career in microbiology! Growing bacteria might be one of the easiest things to do as a scientist. Also, as you’ve probably discovered, it’s even easier to do when you’re trying to prevent bacteria from growing where it shouldn’t be!! When we go…

World of Microbes Part 4: Food and Biofuels.

World of Microbes Part 4: Food and Biofuels.

In this World of Microbes series, so far we’ve covered the importance of microbes in medicine including their use in vaccines, antibiotics, probiotics and protein production of antibodies for immunotherapy. Now we’ll diverge away from medicine and explore two other important fields where microbes are invaluable – food production and biofuel. Food Production When you…

Mastering the Art of Growing THP-1 cells

Mastering the Art of Growing THP-1 cells

Tissue culture can sometimes seem like a black art. Too careful—your cells go down. Not careful enough—your cells go down. A butterfly flutters its wings in the middle of the Atlantic Ocean—your cells go down. It’s annoying, it’s frustrating, and there are times (and I’m speaking from personal experience here) that you’ll end up chucking…

Chromium Release Assay: The Old School Way of Testing Cytotoxic T Cells

Chromium Release Assay: The Old School Way of Testing Cytotoxic T Cells

There are several methods you can use to see if your T cells are cytotoxic, but a chromium release assay using radioactive 51chromium (51Cr) is one of the oldest. It gives good results, and is great for labs that can’t afford or don’t have flow cytometry readily available. Here, I will outline a simple method…

Pre-Analytical Sample Handling: What Can You Do Better?

Pre-Analytical Sample Handling: What Can You Do Better?

If you study human disease, you will likely handle a pre-analytical sample or two (or hundreds).  For example, you could handle whole blood, serum or plasma, tissue biopsies, urine, fecal samples, cerebrospinal fluid, or synovial fluid—to name a few. You will probably use these samples to look for specific metabolites, proteins, or nucleic acids that provide…

Three Steps for Setting up a Drug Screening Assay

Three Steps for Setting up a Drug Screening Assay

An anti-cancer drug or antibody drug conjugate (ADC) screening assay is the first step to establish the utility of a drug candidate in killing cancer cells. Nevertheless, these assays are time consuming and tedious. The purpose of this article is to make things easier when you are required to set up these in vitro screening…

Introducing You to the Wonderful World of Microbes!

Introducing You to the Wonderful World of Microbes!

Welcome to the microbe series where we have a very exciting line-up planned over the coming months. Here we will talk about everything microbial, including the uses of microbes in industry and medicine, emerging pathogens, diagnostics, and much, much more! Let’s kick off this series with an introduction into these wonderful, yet sometimes nasty, organisms…

Four Tips for Working with Human Clinical Samples

Four Tips for Working with Human Clinical Samples

While using human clinical samples in your research can provide robust and heterogeneous results applicable to larger portions of the population, working with these samples presents its own set of challenges. Here are some tricks I have learned to help isolate and grow your cells of interest while eliminating stromal, blood, or other undesired contaminants….

AAV Production Part I: Tips for Setting Up

AAV Production Part I: Tips for Setting Up

What is Adeno-Associated Virus? Adeno-associated virus (AAV) is a popular gene therapy vector. Different AAV serotypes infect different cell and tissue types with varying efficiency, so it’s a good idea to select the serotype most relevant to your work. The most commonly used AAV serotypes are 1, 2, 5, 6, 8, 9 and DJ, although…

How to Overcome Minor Issues in Anchorage-Independent Assays

How to Overcome Minor Issues in Anchorage-Independent Assays

Anchorage-independent assays test the ability of cells to grow independent of a solid surface. The assay is used to check the malignant potential of cancer cells. Cancer researchers generally do this experiment for any kind of confirmation of the oncogenic potential of an oncogene or a tumor suppressor in cancer cells. However, we do encounter…

Sonication – 7 Tips for Mastering the Art

Sonication – 7 Tips for Mastering the Art

Sonication is mostly used during preparation of protein extracts to help break apart the cell. Although most lysis buffers have buckets of detergent that lyse cell membranes, sonication just gives an extra hand in breaking everything apart. Sonication also breaks up, or shears, DNA in a sample—preventing if from interfering with further sample preparation. Have…

How to Extract and Amplify Microalgal Genomic DNA

How to Extract and Amplify Microalgal Genomic DNA

There is a growing trend in using microalgae as the expression system for heterologous proteins. However, I find most protocols dealing with microalgae available online are not that great or informative! So I would like to share my experience in using Chlamydomonas reinhardtii as the model microorganism. Before you start expressing your protein, you need…

Murine Matchmaking: Starting a Breeding Colony of Mice

Murine Matchmaking: Starting a Breeding Colony of Mice

Mice are an extremely powerful research tool, but they do take extra time and effort. While you can purchase mice from vendors like Jackson Labs or outsource your genetic engineering to a number of companies, it’s often necessary, and more economical, to start a breeding colony. Even if you get your mice commercially, you’ll probably…

For the Love of Mouse!  How to Start Working with Mice

For the Love of Mouse! How to Start Working with Mice

Researchers have always been in search of model organisms that can be used to study and explore biological phenomena to make discoveries that can be extrapolated to more complex higher organisms like humans. Of the various model organisms developed and used, starting from the ubiquitous E. coli and S. cerevisiae to the humble D. melanogaster…

The Messy World of Human Tissue Research

The Messy World of Human Tissue Research

In neuroscience and other biomedical research fields, animal models allow us to answer critical but otherwise impossible questions. Despite the value of these models, however, sometimes nothing can replace the real deal—human tissue. The limitations of human tissue research stem from the variability between subjects and the risk for covariates that influence your results. Which…

Open and Closed:  Two Ways to Grow Your Own Algae

Open and Closed: Two Ways to Grow Your Own Algae

In my last article, I talked about the basic protocols and experiments conducted in the process of converting algae into biofuel. Our ability to culture algae has efficiently improved over the years. Continuous improvisation in basic techniques has helped us to understand the growth limiting step of algae culture. In this article, I will discuss…

World of Microbes Part 3: Antibody Production with Microbes

World of Microbes Part 3: Antibody Production with Microbes

When you think of microbes what comes to mind? Moldy bread, Penicillin and antibiotics? Vaccines? Fermented food, like yogurt and kombucha? And the latest Probiotics health craze? How about antibody production for immune therapy? Maybe not so much, but you should know that the use of microbes is wide and ever growing. Now researchers are finding…

The Care and Keeping of Primary Murine B Cells

The Care and Keeping of Primary Murine B Cells

So you want to work with mouse B cells? Primary murine B cells are a difficult, yet fascinating system to work with and can help deepen your understanding of an immunological system. You can study many things with primary B cells, including: immune activation antibody production cell-cell interactions between immune cells and immune phenotype These…

The World of Microbes (II): The Gut Microbiome in Health and Disease

The World of Microbes (II): The Gut Microbiome in Health and Disease

If you google “What’s hot in medicine,” you will see the word “gut microbiome” popping up in every corner of the webpage. Microbes keep your body functioning in balance – from digesting complex carbohydrates to fighting off foreign pathogens and educating your immune system. They have even been linked to maintaining brain function as well….

An Out of Body Experience: ex vivo Tissue Culture for Cancer Drug Screening

An Out of Body Experience: ex vivo Tissue Culture for Cancer Drug Screening

When choosing a model system for culturing tumor cells, we often think of the obvious tried and tested models. In vitro methods include cell lines that have been specifically selected to grow in culture flasks in incubators. While conveniently available, consistent and reproducible, cell lines are limited in that they may not represent the desired…

Beginners Guide to Setting Up Migration and Invasion Assays

Beginners Guide to Setting Up Migration and Invasion Assays

So you have a gene or protein that you think may be involved in migration or invasion and the next step is to embark on migration assays. These assays are useful for testing fundamental migratory processes, such as embryonic development, immune response, metastasis and angiogenesis. For a long time these have been an invaluable mainstay…

Cell Culture is No Longer Flat: Three Dimensional Cell Culture

Cell Culture is No Longer Flat: Three Dimensional Cell Culture

Three dimensional cell culture mimics the extracellular matrix (ECM) that offers the structure and support for cells in vivo, thus creating the complex architecture and network required for cellular communication. For 3D cell culture beginners (or enthusiasts), the information available may seem overwhelming. It sure was for me. But it can be simplified. For example,…

How to Culture Primary Human Bronchial Epithelial Cells

How to Culture Primary Human Bronchial Epithelial Cells

Human bronchial epithelial cells (HBECs) are a challenge to culture. As highly specialised cells that exist in carefully ordered multi-layered structures, they are especially fickle and finding optimum conditions to keep them happy is tricky. The cultures are also extremely sensitive to tiny changes in routine or environment. However, there are certain basic principles that…

Troubleshooting and Optimizing your Mouse Breedings

Troubleshooting and Optimizing your Mouse Breedings

Anyone working with laboratory animals has probably realized that simply putting two animals together does not always yield new offspring and reliable continuity of the animal line – unfortunately animal husbandry isn’t that simple! Of course, apart from making sure that the two animals put together are from different genders, there are a lot of…

Measuring Invasion – Round is the New Flat

Measuring Invasion – Round is the New Flat

I’m sure many of us are aware that the world of cancer research is exploding with the idea of cancer stem cells. This exciting hypothesis suggests that there is a small population of cells in the tumor that have stem cell-like properties. These stem-like cells are able to proliferate and differentiate into all the different…

Practical Secrets of the Bacterial World for the Uninitiated

Practical Secrets of the Bacterial World for the Uninitiated

As a protein biochemist where bacteria were mere workhorses, imagine my surprise when I began work in a bonafide micro lab! I discovered that bacteria could be much fussier than my good ol’ cloning and expression friends E. coli DH5ɑ or BL21. One broth would not do for all, some even required blood! No, no,…

Expanding Possibilities: Why You Need to Look into Viral Transduction

Expanding Possibilities: Why You Need to Look into Viral Transduction

We have already looked into the different types of viral expression systems and when you might use one over another in my previous article. But why would you use viral transduction over similar techniques like plasmids? Just a reminder: Transfection is a lab technique where nucleic acids or proteins may be introduced into cells. When…

Want to Increase Your Lentiviral Titers?  Focus on Your 293T Cells

Want to Increase Your Lentiviral Titers? Focus on Your 293T Cells

Producing lentiviral or retroviral vectors is theoretically fairly straightforward. However, anyone new to viral vector work is usually confronted with vast amounts of confusing information. It seems that anyone who has ever made a lentivirus has their own protocols and is adamant that their method is the best one to follow. In reality, there are…

The 31 Flavors of Drosophila Electrophysiology Recording Solutions

The 31 Flavors of Drosophila Electrophysiology Recording Solutions

If you want to know what is going on in the brain of drosophila you can use neurobiology imaging techniques to get a global whole-brain perspective. However, such techniques are slow compared to the rapid nature of the neuronal electrical activity, which may be better studied using Drosophila electrophysiology. Lab techniques are often shrouded in…