Cell Culture and Transfection

Your Media Choice Might Be Designing Your T-Cell Fate

Posted on by Simon Moe

Why Metabolism Matters in T-Cell Expansion

Adoptive T-cell therapies rely on generating metabolically fit, functional cells during ex vivo expansion—but this process often pushes T cells toward highly glycolytic, terminally differentiated states that limit their persistence and therapeutic potential. These metabolic programs begin shifting within hours of activation, therefore understanding early metabolic remodeling is essential for designing culture conditions that support durable, cytotoxic, and memory-enriched T-cell populations.

Researchers at Promega set out to address this challenge by systematically mapping how media composition and activation strength shape T-cell metabolism during the first 72 hours after stimulation. Using a suite of bioluminescent assays, they profiled intracellular energy cofactors, redox balance, and extracellular metabolites across several conditions. This approach revealed distinct, media-driven metabolic states that not only emerged early but also predicted downstream expansion, proliferation, and cytotoxic function.

Their work demonstrates how integrating metabolic profiling into in vitro expansion workflows can provide a more informed framework for optimizing T-cell manufacturing strategies.

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Why mRNA Transfection Is Transforming Transient Expression Workflows

Posted on by Shannon Sindermann

Transfection is a core technique in molecular biology used to introduce foreign nucleic acids—such as DNA, RNA, or small RNAs like siRNA, shRNA, and miRNA—into eukaryotic cells. This enables researchers to manipulate gene expression and study cellular processes, disease mechanisms and therapeutic strategies (1).

Advances in transfection technology now support a range of nucleic acid types and cell models. Researchers can pursue transient or stable expression to achieve specific goals: knocking down transcripts, expressing proteins, or probing promoter activity in systems from immortalized lines to stem cells (1).

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What 32,000 3D Spheroids Revealed About Culture Conditions

Posted on by Shannon Sindermann
3D Spheroid Cell Culture

Three-dimensional (3D) cell culture systems have become essential tools in cancer research, drug screening and tissue engineering—offering a more physiologically relevant alternative to traditional 2D cultures, which often fail to replicate key in vivo microenvironment features. But as the field has evolved, variability in experimental outcomes has become a key challenge, limiting their reproducibility and translation into clinical settings. While spheroids offer layered architecture, nutrient gradients and multicellular interactions, inconsistent culture methods have made it difficult to draw reliable conclusions across labs.

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Small RNA Transfection: How Small Players Can Make a Big Impact

Posted on by Simon Moe

When looking at small aspects of living things, especially cells, it can often be difficult to fully grasp the magnitude of regulation employed within them. We first learn the central dogma in high school biology. This is the core concept that DNA makes RNA and RNA makes protein. Despite this early education, it can be lost on many the biological methods that are employed to regulate this process. This regulation is very important when one considers the disastrous things that can occur when this process goes askew, such as cancer, or dysregulated cell death. Therefor it is very important to understand how these regulatory mechanisms work and employ tools to better understand them.

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Insects and Science: Optimizing Work with Sf9 Insect Cells

Posted on by Shannon Sindermann

Insects are a keystone species in the animal kingdom, often providing invaluable benefits to terrestrial ecosystems and useful services to mankind. While many of them are seen as pests (think mosquitos), others are important for pollination, waste management, and even scientific research.

Insect biotechnology, or the use of insect-derived molecules and cells to develop products, is applied in a diverse set of scientific fields including agricultural, industrial, and medical biotechnology. Insect cells have been central to many scientific advances, being utilized in recombinant protein, baculovirus, and vaccine and viral pesticide production, among other applications (5).

Therefore, as the use of insect cells becomes more widespread, understanding how they are produced, their research applications, and the scientific products that can be used with them is crucial to fostering further scientific advancements.

Primary Cell Cultures and Cell Lines

Cell culture - Cell lines - Insect Cells

In general, experimentation with individual cells, rather than full animal models, is advantageous due to improved reproducibility, decreased space requirements, less ethical concerns, and a reduction in expense. This makes primary cell cultures and cell lines essential contributors to basic scientific research.

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I Have My Luciferase Vector, Now What?

Posted on by Joliene Lindholm
Choosing and Optimizing Transfection Methods

Here in Technical Services we often talk with researchers at the beginning of their project about how to carefully design and get started with their experiments. It is exciting when you have selected the Luciferase Reporter Vector(s) that will best suit your needs; you are going to make luminescent cells! But, how do you pick the best way to get the vector into your cells to express the reporter? What transfection reagent/method will work best for your cell type and experiment? Do you want to do transient (short-term) transfections, or are you going to establish a stable cell line?

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How to Reduce Cell Culture Variability

Posted on by Johanna Lee

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.

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The Cell Line Identity Crisis

Posted on by Nicole Sandler
29981164-Whistle

If you work with cell lines you may have paid attention to the dramatic headline published last month in the online journal STAT, Thousands of studies used the wrong cells, and journals are doing nothing.” In their column The Watchdogs (“Keeping an eye on misconduct, fraud, and scientific integrity”), Ivan Oransky and Adam Marcus call out the fact that scientists continue to publish research using cell lines that are contaminated or misidentified. Recent estimates have found that the percentage of misidentified cell lines used by scientists is as high as 20 to 36. The blame here is being placed on the peer reviewed journals for not blowing the whistle. The authors call for journals to put some “kind of disclaimer on the thousands of studies affected.”

This is not a new claim. The continuing problem of cell line misidentification, of lack of authentication, has been covered before in various channels. It’s easy to find news publicizing yet another retracted publication. In May 2015 the journal Nature required authors of all submitted manuscripts to confirm the identity of cell lines used in their studies and provide details about the source and testing of their cell lines.

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Improving the Success of Your Transfection

Posted on by Sara Klink
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Not every lab has a tried and true transfection protocol that can be used by all lab members. Few researchers will use the same cell type and same construct to generate data. Many times, a scientist may need to transfect different constructs or even different molecules (e.g., short-interfering RNA [siRNA]) into the same cell line, or test a single construct in different cultured cell lines. One construct could be easily transfected into several different cell lines or a transfection protocol may work for several different constructs. However, some cells like primary cells can be difficult to transfect and some nucleic acids will need to be optimized for successful transfection. Here are some tips that may help you improve your transfection success.

Transfect healthy, actively dividing cells at a consistent cell density. Cells should be at a low passage number and 50–80% confluent when transfected. Using the same cell density reduces variability for replicates. Keep cells Mycoplasma-free to ensure optimal growth.

Transfect using high-quality DNA. Transfection-quality DNA is free from protein, RNA and chemical contamination with an A260/A280 ratio of 1.7–1.9. Prepare purified DNA in sterile water or TE buffer at a final concentration of 0.2–1mg/ml.

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General Considerations for Transfection

Posted on by Sara Klink

Many studies, from reporter assays to protein localization to BRET and FRET, require successful transfection first. Yet, transfection can be tricky and difficult. There are many considerations when planning transfection of your cells including reagent selection, stable or transient experiment, type of molecule and endpoint assay used. Here we discuss these considerations to help you plan a successful transfection scheme for your experimental system. Continue reading “General Considerations for Transfection”