Maximize Your Time in the Lab: Improve Experimental Reproducibility with Thaw-and-Use Cells

Many cell biology researchers can name their department’s  or institutions’s “cell culture wizard”—the technician with 20+ years of experience whose cell cultures are always free from contamination, exhibit reliable doubling rates and show no phenotype or genotype weirdness. Cell culture takes skill and experience. Primary cell culture can be even more difficult still, and many research and pharmaceutical applications require primary cells.

Yet, among the many causes of failure to replicate study results, variability in cell culture stands out (1). Add to the normal challenges of cell culture a pandemic that shut down cell culture facilities and still limits when and how often researchers can monitor their cell culture lines, and the problem of cell culture variability is magnified further.

Treating Cells as Reagents

A good way to reduce variability in cell-based studies is to use the thaw-and-use frozen stock approach. This involves freezing a large batch of “stock” cells, then performing quality control tests to ensure they respond appropriately to treatment. Then whenever you need to perform an assay, just thaw another vial of cells from that batch and begin your assay—just like an assay reagent! This approach eliminates the need to grow your cells to a specific stage, which could take days and introduce more variability.

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How to Reduce Cell Culture Variability

Scenario 1: Jake needs a flask of MCF-7 cells for an assay, so he sends an email to the graduate student listserv asking for cells. Melissa replies that she has an extra flask of cells that she could share. Jake happily accepts the cells and begins his experiment.

Scenario 2: Michael passaged his cells yesterday and, according to the protocol, was supposed to plate cells today for treatment. However, his previous experiments were delayed, so he decides to plate them tomorrow instead. The cells look healthy, so it should be ok.

What is wrong with the above scenarios? These actions may seem harmless, but they could be the cause of variability, leading to irreproducible results. Continue reading “How to Reduce Cell Culture Variability”

Practical Tips for HEK293 Cell Culture When Using cAMP-Glo™ Assay

HEK293 cells stably expressing HaloTag®-ECS (ExtraCellular Surface; comprised of a signal sequence and single transmembrane domain of β1-integrin) fusion protein labeled with HaloTag® Alexa Fluor® 488 Ligand and then imaged.
HEK293 cells stably expressing HaloTag®-ECS (ExtraCellular Surface; comprised of a
signal sequence and single transmembrane domain of β1-integrin) fusion protein labeled
with HaloTag® Alexa Fluor® 488 Ligand and then imaged.
G Protein Coupled Receptors represent one of the largest classes of cell surface receptors and one of the most important classes for drug targets. Fifty of the top 200 drugs target GPCRs. GPCRs respond to various stimuli like light, odors, hormones, neurotransmitters and others. They cover virtually all therapeutic areas. When a particular GPCR is implicated in a disease, researchers screen the GPCR and its signaling pathways, the hope being that promising therapeutic targets might be identified. Major G-protein families signal via secondary messengers like cAMP, which in turn activate a range of effector systems to change cell behavior and/or gene transcription. There are various approaches and methods to study GPCRs and measure the increase or decrease of intracellular cAMP. However, the fastest and the most sensitive among all methods is a plate based cAMP-Glo™ Assay. Continue reading “Practical Tips for HEK293 Cell Culture When Using cAMP-Glo™ Assay”

Preventing the Heartache of Cell Line Misidentification

Golden maskIt’s a scientist’s nightmare: Spending time and resources to investigate a biological phenomenon only to learn later that your cells are not what you think they are—their true identities hidden. As a result, all of the data that you’ve generated with those cells, published and unpublished, are cast into doubt. You thought that you knew your cells, that you could trust them, but your trust was misplaced. At some point, perhaps even before the traitorous cell line entered your laboratory, the cells were mislabeled, misidentified or contaminated with another cell line. It didn’t have to be this way. There are easy steps you can take to prevent the headache and heartache of cell line misidentification and contamination.

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Considerations for Successful Cell-Based Assays I: Choosing Your Cells

For those of us entering the world of cell-based assays from a classical or molecular genetics background, the world of cell culture can be daunting. Yet to truly understand how the genetic mutation behind a particular phenotype works, we need to look at the biochemistry and cell biology where it all occurs: the cell.

This series of blogs will cover several topics to consider when designing your cell-based assays. In this first installment, we discuss the basics of choosing the cell type for your assay. Continue reading “Considerations for Successful Cell-Based Assays I: Choosing Your Cells”

Sloppy Technicians and the Progress of Science

Then, in 1953, a geneticist in Texas accidentally mixed the wrong liquid with HeLa and a few other cells, and it turned out to be a fortunate mistake. The chromosomes inside the cells swelled and spread out, and for the first time, scientists could see each of them clearly. —Rebecca Skloot, The Immortal Life of Henrietta Lacks

Okay, Ms. Skloot, no fair teasing a geneticist reader like that. Who was the scientist in Texas? What was the wrong liquid? How long did it take for the scientist to realize he had launched the entire field of cytogenetics with his mistake? This inquiring mind wants to know. Continue reading “Sloppy Technicians and the Progress of Science”

Cells, Specificity and the Seven-Year-Old Brain

We would all agree that being specific is important.  But sometimes what it is important to be specific about is not so easy to agree upon.

My seven-year-old son is a good example. Continue reading “Cells, Specificity and the Seven-Year-Old Brain”