Cell Biology

RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5– and serine 2–phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription.

Graphical overview

The sirtuin 6 has emerged as a regulator of acute and chronic immune responses. Recent findings show that SIRT6 is necessary for mounting an active inflammatory response in macrophages. In vitro studies revealed that SIRT6 is stabilized in the cytoplasm to promote tumor necrosis factor (TNFα) secretion. Notably, SIRT6 also promotes TNFα secretion by resident peritoneal macrophages upon lipopolysaccharide (LPS) stimulation in vivo. Although many studies have investigated SIRT6 function in the immune response through different genetic and pharmacological approaches, direct measurements of in vivo SIRT6 expression in immune cells by flow cytometry have not yet been performed. Here, we describe a step-by-step protocol for peritoneal fluid extraction, isolation, and preparation of peritoneal cavity cells, intracellular SIRT6 staining, and flow cytometry analysis to measure SIRT6 levels in mice peritoneal macrophages. By providing a robust method to quantify SIRT6 levels in different populations of macrophages, this method will contribute to deepening our understanding of the role of SIRT6 in immunity, as well as in other cellular processes regulated by SIRT6.

Graphical abstract:

DNA double strand breaks (DSBs) constantly arise in cells during normal cellular processes or upon exposure to genotoxic agents, and are repaired mostly by homologous recombination (HR) and non-homologous end joining (NHEJ). One key determinant of DNA DSB repair pathway choice is the processing of broken DNA ends to generate single strand DNA (ssDNA) overhangs, a process termed DNA resection. The generation of ssDNA overhangs commits DSB repair through HR and inhibits NHEJ. Therefore, DNA resection must be carefully regulated to avoid mis-repaired or persistent DSBs. Accordingly, many approaches have been developed to monitor ssDNA generation in cells to investigate genes and pathways that regulate DNA resection. Here we describe a flow cytometric approach measuring the levels of replication protein A (RPA) complex, a high affinity ssDNA binding complex composed of three subunits (RPA70, RPA32, and RPA14 in mammals), on chromatin after DNA DSB induction to assay DNA resection. This flow cytometric assay requires only conventional flow cytometers and can easily be scaled up to analyze a large number of samples or even for genetic screens of pooled mutants on a genome-wide scale. We adopt this assay in G₀- and G₁- phase synchronized cells where DNA resection needs to be kept in check to allow normal NHEJ.

Cyanobacteria are Gram-negative oxygen-producing photosynthetic bacteria that are useful in the pharmaceutical and biofuel industries. Monitoring of oxidative stress under fluctuating environmental conditions is important for determining the fitness, survival, and growth of cyanobacteria in the laboratory as well as in large scale cultivation systems. Here, we provide a protocol developed using unicellular Synechococcus elongatus PCC 7942 and filamentous Fremyella diplosiphon BK14 cyanobacteria for high-throughput oxidative stress measurement by 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH-DA) and flow cytometry (FCM). We also provide details for the optimization of cell number, dye concentration, and FCM parameters for each organism before it can be utilized to quantify reactive oxygen species (ROS). FCM-based method can be used to measure ROS in a large population of cyanobacterial cells in a high-throughput manner.

Graphical abstract:

Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the differentiation of primary AML cells, thus allowing for mechanistic studies in vitro. Additionally, the co-culture on OP9-M2 stromal is superior in preserving surface marker expression of primary (adult and pediatric) AML cells in comparison to stroma-free culture. Thus, by combining the co-culture with multicolor, high-throughput FACS, we can evaluate the effect of hundreds of small molecules on multi-parametric processes including: cell survival, stemness (leukemic stem cells), and myeloid differentiation on the primary AML cells at a single-cell level. This method streamlines the identification of potential therapeutic agents, but also facilitates combinatorial screening aiming, for instance, at dissecting the regulatory pathways in a patient-specific manner.

Graphic abstract:

Schematic representation of the ex vivo small molecule screening of primary human acute myeloid leukemia.
Irradiated, sub-confluent OP9-M2 stromal cells are plated in half-area 96 wells plates 4–16 h prior to adding primary AML cells. Compounds are added 36–48 h later and effects on cell number, leukemic stem cell population, and myeloid differentiation are quantifed by FACS after 4 days of treatment.

All eukaryotic cells are equipped with transmembrane lipid transporters, which are key players in membrane lipid asymmetry, vesicular trafficking, and membrane fusion. The link between mutations in these transporters and disease in humans highlights their essential role in cell homeostasis. Yet, many key features of their activities, their substrate specificity, and their regulation remain to be elucidated. Here, we describe an optimized quantitative flow cytometry-based lipid uptake assay utilizing nitrobenzoxadiazolyl (NBD) fluorescent lipids to study lipid internalization in mammalian cell lines, which allows characterizing lipid transporter activities at the plasma membrane. This approach allows for a rapid analysis of large cell populations, thereby greatly reducing sampling variability. The protocol can be applied to study a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane, and to uncover the dynamics of lipid transport at the plasma membrane.

Graphic abstract:

Internalization of NBD-labeled lipids from the plasma membrane of CHO-K1 cells.

Planarians are free-living flatworms that emerged as a crucial model system to understand regeneration and stem cell biology. The ability to purify neoblasts, the adult stem cell population of planaria, through fluorescence-activated cell sorting (FACS) has tremendously increased our understanding of pluripotency, specialization, and heterogeneity. To date, the FACS-based purification methods for neoblasts relied on nuclear dyes that discriminate proliferating cells (>2N), as neoblasts are the only dividing somatic cells. However, this method does not distinguish the functional states within the neoblast population. Our work has shown that among the neoblasts, the pluripotent stem cells (PSCs) are associated with low mitochondrial content and this property could be leveraged for purification of the PSC-enriched population. Using the mitochondrial dye MitoTracker Green (MTG) and the nuclear dye SiR-DNA, we have described a method for isolation of PSCs that are viable and compatible with downstream experiments, such as transplantation and cell culture. In this protocol, we provide a detailed description for sample preparation and FACS gating for neoblast isolation in planaria.

Natural killer (NK) cells are large granular lymphocytes that keep in check the health of neighboring cells through a large array of intrinsically expressed germline-coded receptors. Most importantly, CD16 is a low affinity Fc receptor for IgG that mediates the antibody-dependent cellular cytotoxicity (ADCC) of NK cells, bridging the innate and adaptive immunities. There has been a significant interest in genetically engineering NK cells to enhance its ADCC, with the ultimate goal to produce off-the-shelf NK cell therapy products that can be combined with target-specific monoclonal antibodies to improve clinical outcomes. Previous protocols of ADCC assays use complex cell-based antigen-antibody models, which are both costly and time-consuming. This current protocol is devoid of target cells and uses plate-bound immobilized anti-CD16 antibodies as the trigger. It greatly shortens the experimental time, while faithfully evaluating NK cells ADCC.

Graphic abstract:

Workflow of stimulating NK cells via CD16 by plate-bound anti-CD16 mAb.

Regulatory T cells (Tregs) suppress immune responses via a variety of mechanisms and can be used as a cellular therapy to induce tolerance. The function of Tregs is commonly assessed in vitro using assays that measure suppression of effector T cell proliferation and/or cytokine production. However, Tregs can also suppress the function of antigen presenting cells, creating a need for methodology to routinely measure this aspect of their function. This protocol describes a method to measure human Treg-mediated suppression of CD80 and CD86 expression on mature, monocyte-derived dendritic cells. Representative data show suppression mediated by polyclonal Tregs as well as antigen-specific Tregs generated using chimeric antigen receptor (CAR) technology. This method can be used in parallel to T cell suppression assays to measure the functional activity of human Tregs.

The relapsing malaria species, Plasmodium vivax, is the most widely distributed and difficult-to-treat cause of human malaria. The merozoites of P. vivax preferentially invade ephemeral human CD71⁺ reticulocytes (nascent reticulocytes), thereby limiting the development of a robust continuous culture in vitro. Fortunately, P. vivax’s sister species, P. cynomolgi Berok, can be cultured continuously, providing the ability to screen novel therapeutics drug and vaccine candidates in a reliable and high-throughput manner. Based on well-established growth inhibition activity (GIA) assays against P. falciparum and P. knowlesi, this protocol adopts the current flow cytometry assay methodology and investigates P. vivax inhibitory antibodies using the P. cynomolgi Berok invasion model based on the thiol-reactivity and DNA abundance of viable parasites in macaque erythrocytes. Established GIA assays screen antibodies at either a single concentration or high/low dose concentrations to provide quick insights for prioritizing potential antibodies capable of specifically interrupting parasite ligand and host receptor binding with minimal concentrations. Hence, this protocol expands on the existing GIA assay by using serially diluted antibodies and generating a dose-response curve to better quantify the inhibitory efficacy amongst selected vaccine candidates.