Extracellular vesicles (EVs) secreted by all cell types play crucial roles in intercellular communication through the transfer of bioactive molecules, including proteins, lipids, and nucleic acids, within tissues and thr...Extracellular vesicles (EVs) secreted by all cell types play crucial roles in intercellular communication through the transfer of bioactive molecules, including proteins, lipids, and nucleic acids, within tissues and throughout the organism. In cutaneous biology, EVs have emerged as key mediators in maintaining skin homeostasis and regulating pathological processes. However, obtaining sufficient quantities of EVs from primary cell cultures remains technically challenging due to inherent limitations in cell number, proliferative capacity, and EV production efficiency compared to immortalized cell lines. In this chapter, we present detailed, step-by-step protocols specifically optimized for isolating small EVs from primary human dermal fibroblasts and epidermal keratinocytes. These methodologies consistently yield sufficient EV quantities for comprehensive functional studies.
The generation of epithelial equivalents at air-liquid interface on commercially available membrane inserts is currently a routine procedure. However, producing similar equivalents on voluminous polymeric scaffolds, espe...The generation of epithelial equivalents at air-liquid interface on commercially available membrane inserts is currently a routine procedure. However, producing similar equivalents on voluminous polymeric scaffolds, especially those made from novel materials, can be challenging, as newly developed materials are often difficult to maintain. An adequate culture system design may be of decisive importance for such an approach. Here, we describe the generation/growing of epidermal equivalents on nonwoven (electro-spun) scaffolds that are getting increasing attention and application in skin bioengineering.
Three-dimensional culturing of human keratinocytes at the air-liquid interface yields a fully stratified epidermis, including a functional stratum corneum, and thus enables the study of epidermal structure and function i...Three-dimensional culturing of human keratinocytes at the air-liquid interface yields a fully stratified epidermis, including a functional stratum corneum, and thus enables the study of epidermal structure and function in the context of biomedical, toxicological, and pharmaceutical sciences. Here, we provide an updated step-by-step, detailed protocol for the isolation of human primary keratinocytes and the development of human epidermal equivalents (HEEs) generated from primary keratinocytes or immortalized keratinocytes (N/TERT-1; N/TERT-2G). This chapter includes information on widely accepted procedures for the analysis of epidermal equivalent tissue morphology, keratinocyte proliferation and differentiation, and gene expression. In addition, this chapter handles qualitative and quantitative procedures for measuring epidermal barrier function, including the more recently developed electrical impedance spectroscopy (EIS).
Here, we describe the isolation of epidermal melanocytes and hair follicle melanocytes from human skin tissue. Epidermal and hair follicle melanocytes are two distinct populations of melanocytes that are contained within...Here, we describe the isolation of epidermal melanocytes and hair follicle melanocytes from human skin tissue. Epidermal and hair follicle melanocytes are two distinct populations of melanocytes that are contained within the skin and the hair follicle, with differing yet overlapping roles. Epidermal melanocytes are normally isolated from the epidermis of vellus-haired skin tissue, for example, face, breast, abdomen, while hair follicle melanocytes are derived from the outer root sheath (ORS) of the middle/lower terminal anagen hair follicles of the scalp. These methods utilize ethically sourced human skin tissue obtained from donors undergoing plastic surgery.
The establishment of primary cells from fresh tissue is a widely used method for investigating human tissue in vitro. The skin harbors different cell populations in the dermis and the hair follicle, which can be isolated...The establishment of primary cells from fresh tissue is a widely used method for investigating human tissue in vitro. The skin harbors different cell populations in the dermis and the hair follicle, which can be isolated for downstream analysis. Here, we describe the isolation of four dermal fibroblast populations from human haired skin and their maintenance in culture. The four cell population isolations described are the papillary dermal fibroblast cells, the reticular dermal fibroblast cells, the hair follicle dermal sheath cells, and the hair follicle dermal papilla cells.
The migration of epidermal keratinocytes is the basis for skin re-epithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward an...The migration of epidermal keratinocytes is the basis for skin re-epithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward and effective method to assess their migratory capacity. The mechanical scratch of a confluent monolayer directly disrupts the adhesion of the keratinocytes to one another and to the underlying matrix, resembling the physical trauma of a wound in an in vitro assay. The keratinocytes will undergo an epithelial-to-mesenchymal transition, which will confer an ability to migrate toward each other to cover the gap by restructuring cell-cell and cell-extracellular matrix connections. However, a good scratch-wound method and protocol to ensure scratch reproducibility is essential, particularly when using primary cell cultures where donor variability may also impact results.
The use of zebrafish in research is becoming increasingly popular, requiring the development of analytical techniques to suit the organism. One of these techniques is immunofluorescence staining, which allows the visuali...The use of zebrafish in research is becoming increasingly popular, requiring the development of analytical techniques to suit the organism. One of these techniques is immunofluorescence staining, which allows the visualization and quantification of specific proteins of interest. The immunofluorescence staining technique involves the binding of primary and secondary antibodies (with attached fluorophores) to a protein of interest-in this case, macrophage migration inhibitory factor (MIF). Due to the small size and transparency of zebrafish larvae, this can be performed using the whole body, so that no sectioning equipment is required. Making the technique even more adaptable and user-friendly, the high genetic homology observed between humans and zebrafish often allows the application of antibodies raised against human antigens in zebrafish imaging. For example, there is a 79% amino acid sequence homology between human and zebrafish MIF, with previous imaging demonstrating clear similarities in the expression patterns between the two species. This chapter will provide an in-depth description of the methods involved in immunofluorescence staining of MIF in zebrafish larvae, using tailfin transection as the experimental, pro-inflammatory insult.
In vitro co-culture models of human cells and microbial pathogens offer a controlled system to study host-pathogen interactions. This chapter details the methods to establish such co-culture models for a fungal and an am...In vitro co-culture models of human cells and microbial pathogens offer a controlled system to study host-pathogen interactions. This chapter details the methods to establish such co-culture models for a fungal and an amoebic infection using two different human cell lines. Employing this experimental system allows the researcher to evaluate MIF's contribution to infection-driven host cell death and pro-inflammatory gene expression.
The primary cilium, a microtubule-based organelle that protrudes from the surface of most eukaryotic cell types, serves as a signaling hub, playing a vital role in development and maintaining tissue homeostasis. Given th...The primary cilium, a microtubule-based organelle that protrudes from the surface of most eukaryotic cell types, serves as a signaling hub, playing a vital role in development and maintaining tissue homeostasis. Given the ever-increasing significance of primary cilia in both health and disease, discovering new factors involved in ciliogenesis will enhance our comprehension of the role of this organelle. The macrophage migration inhibitory factor (MIF) has been acknowledged as a secreted cytokine involved in the pathogenesis of various human diseases. MIF is expressed and released ubiquitously by numerous cell types and tissues, and unlike other cytokines, intracellular MIF exhibits unique functional characteristics. In a recent study, it was reported that MIF is a significant contributor to cilia biogenesis and serves as a novel transcriptional regulator in homeostasis. Utilizing immunofluorescent staining and three-dimensional structured illumination microscopy (3D-SIM), which allows the visualization of cellular components with high specificity, the authors demonstrated that MIF is localized and forms a ring-like structure at the proximal end of the centrioles, where it influences cilia biogenesis by affecting the recruitment and accumulation of basal body proteins, including TTBK2, CP110, and CEP290. Here, we describe protocols and optimization tips on how to use standard labeling techniques with 3D-SIM to investigate the role of MIF in primary cilia in both human- and murine-derived ciliated cells. The utilization of 3D-SIM alongside standard staining techniques, while concurrently identifying MIF within the three-dimensional centrosomal structure, presents intriguing new opportunities for MIF in molecular cell biology.
Transfection is the introduction of foreign genetic material (e.g., DNA) into eukaryotic cells with the downstream goal of the expression and characterization of a protein with a specific biological function. This experi...Transfection is the introduction of foreign genetic material (e.g., DNA) into eukaryotic cells with the downstream goal of the expression and characterization of a protein with a specific biological function. This experimental technique is particularly useful in cancer research as it permits the study of oncogenes. When the foreign genetic material is incorporated into the genome of the host cell, then the technique is called "Stable Transfection." Once the transfected DNA has been integrated, the gene of interest is permanently expressed, allowing for research to be conducted over extended periods of time without disruption. Here, we describe the lipid-based stable transfection of CD74 gene variants into the A549 lung adenocarcinoma cell line. CD74 is a type II cell surface receptor with a particular interest in cancer biology due to its key functional role in both solid and hematological malignancies. Our protocol provides a highly reproducible, step-by-step procedure for stable transfection that can be applied to a variety of genes.
Small interfering RNA (siRNA)-mediated depletion of macrophage migration inhibitory factor (MIF) in cell culture systems is essential for understanding the function or relevance of MIF in various cell types under differe...Small interfering RNA (siRNA)-mediated depletion of macrophage migration inhibitory factor (MIF) in cell culture systems is essential for understanding the function or relevance of MIF in various cell types under different experimental conditions. In this article, we provide a detailed description of the methods for siRNA transfection in a terminally differentiated adherent cell line derived from human corneal epithelial cells. Furthermore, we discuss siRNA transfection techniques for two suspension cell lines of monocytic and promyelocytic origin, which can be differentiated into adherent macrophage lines and suspension neutrophil lines, respectively. Following siRNA transfection, we evaluated MIF knockdown at the transcript level through RNA extraction, cDNA synthesis, and real-time PCR analysis.
Inflammasome activation and signaling are hallmarks of immune activation and cellular response. Understanding the complex mechanisms involved in the formation of the NLRP3 inflammasome is vital to accurately evaluating p...Inflammasome activation and signaling are hallmarks of immune activation and cellular response. Understanding the complex mechanisms involved in the formation of the NLRP3 inflammasome is vital to accurately evaluating potentially anti-inflammatory molecules. The oligomerization of ASC is a well-documented indicator of inflammasome activation, and ASC-GFP allows the visualization of these "specks." Here, we detail a higher throughput methodology for monitoring inflammasome activation and screening potential anti-inflammatory compounds utilizing an Incucyte® live cell imager. It provides a platform to observe cell activation and cell death in real-time. Cells can be visualized from initial plating through to inflammasome-induced cell death, granting insights into inflammasome activation kinetics and previously unknown toxicities.
Nuclear factor-κB (NF-κB) is a family of transcription factors that regulate cellular responses to a variety of stimuli, including stress, ultraviolet radiation, microbial components, growth factors, and cytokines. Asses...Nuclear factor-κB (NF-κB) is a family of transcription factors that regulate cellular responses to a variety of stimuli, including stress, ultraviolet radiation, microbial components, growth factors, and cytokines. Assessment of NF-κB activation can be investigated by measuring the expression of NF-κB-responsive genes and their products, typically using polymerase chain reaction (PCR) and/or immunoblotting for protein. Alternatively, translocation of NF-κB subunits from the cytosol to the nucleus can be measured using microscopy techniques. However, these methods can be complex and time-consuming. Luciferase-based reporter systems allow for rapid detection of gene expression. This chapter details the use of myeloid cells stably transfected with NF-κB-inducible luciferase reporter constructs to assess the effects of inflammatory stimuli and MIF inhibitors on NF-κB activation.
The isolation of bone marrow-derived macrophages (BMDMs) from the bones of mice provides an excellent tool to study the different activation states of macrophages both in vitro and ex vivo. This chapter utilizes BMDMs de...The isolation of bone marrow-derived macrophages (BMDMs) from the bones of mice provides an excellent tool to study the different activation states of macrophages both in vitro and ex vivo. This chapter utilizes BMDMs derived from wild-type and -794 CATT7 human transgenic mice to investigate the influence of high human MIF expression on macrophage polarization. Real-time qPCR and ELISA can be used to easily distinguish M1 and M2 polarization phenotypes.
Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine and atypical chemokine that regulates both innate and adaptive immunity. Its broad expression and stress-induced release make MIF a key...Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine and atypical chemokine that regulates both innate and adaptive immunity. Its broad expression and stress-induced release make MIF a key determinant of inflammation, cardiovascular diseases, and cancer. MIF acts as an upstream regulator within a complex ligand-receptor network comprising the more recently discovered MIF paralog, D-dopachrome tautomerase (D-DT, MIF-2), its cognate receptor CD74, and the chemokine receptors CXCR2, CXCR4, and ACKR3/CXCR7. MIF-driven effects, including leukocyte recruitment, cell migration, and inflammatory signaling, largely depend on the specific MIF receptor repertoire of the involved cell types, which can dynamically change in inflammatory settings. Here, we describe an optimized protocol for the ibidi 3D µ-Slide chemotaxis assay to study MIF-mediated pro-migratory effects on human neutrophils, a prototype of innate immune cells, and CD4 T cells, a cornerstone of adaptive immunity. Additionally, we discuss the application of current inhibitor strategies to selectively target and identify the receptor pathways involved.
Immunohistochemistry (IHC) staining is a technique that exploits the specificity of antibodies to detect antigens (proteins) in tissue samples. This usually involves a tissue fixation step to prevent elution, degradation...Immunohistochemistry (IHC) staining is a technique that exploits the specificity of antibodies to detect antigens (proteins) in tissue samples. This usually involves a tissue fixation step to prevent elution, degradation, or modification of the protein of interest. However, the fixation step denatures the protein, altering the characteristic structure. This is particularly problematic when detecting oxidized macrophage migration inhibitory factor (oxMIF), the pathogenic isoform of macrophage migration inhibitory factor (MIF). In the reduced state of MIF, the oxMIF antibody epitopes are hidden within the central pore of the trimeric structure and are thus inaccessible to antibodies. OxMIF is formed through an oxidative post-translational modification of MIF in the pro-oxidative environment of inflammatory processes, as well as in tumors. This oxidation leads to a change in the native conformation of MIF, exposing these epitopes and allowing the detection of oxMIF with anti-oxMIF antibodies. Therefore, sample preparation must not artificially induce structural changes to MIF to enable specific staining of oxMIF. Here, we describe a method for detecting oxMIF in cancer tissue using IHC with oxMIF-specific antibodies that exclude cross-reactivity with reduced MIF.
The enzyme-linked immunosorbent assay (ELISA) is an immunological biochemical assay used to detect and quantify proteins in various sample types including serum, plasma, cell culture medium, cleared bacterial lysate, and...The enzyme-linked immunosorbent assay (ELISA) is an immunological biochemical assay used to detect and quantify proteins in various sample types including serum, plasma, cell culture medium, cleared bacterial lysate, and buffer. To detect the antigen of interest in these samples, specific antibodies targeting accessible epitopes on antigen's surface are employed. Highly affine antibodies with excellent specificity enable accurate and precise detection and quantification of the protein of interest. This high affinity also facilitates the detection of trace amounts of proteins, which is crucial for analyzing cytokines typically present in much lower concentrations compared to plasma proteins. Plasma levels of oxidized macrophage migration inhibitory factor (oxMIF) can reach 150 ng/mL or higher, but concentrations below 20 ng/mL are more common. In addition, differentiating between the pathological isoform oxMIF and MIF is necessary. Here, we describe a method to detect oxMIF and MIF in various sample matrices of biological origin using the sandwich ELISA method.
Macrophage migration inhibitory factor (MIF) exhibits nuclease activity critical in DNA damage pathways linked to neurodegenerative diseases and cancer. Here, we describe dual approaches for measuring this nuclease activ...Macrophage migration inhibitory factor (MIF) exhibits nuclease activity critical in DNA damage pathways linked to neurodegenerative diseases and cancer. Here, we describe dual approaches for measuring this nuclease activity, combining biochemical and cellular analysis. Recombinant MIF is subjected to enzymatic assays with L1 DNA substrates, analyzing cleavage patterns via agarose gel electrophoresis and time-dependent kinetics over 5-30 min. SH-SY5Y cells treated with the DNA-damaging agent MNNG, with or without the MIF nuclease inhibitor PAANIB-1, undergo pulse-field gel electrophoresis to assess DNA fragmentation following cellular stress. These assays can be used to test MIF nuclease activity and screen MIF nuclease activity inhibitors.
Human macrophage migration inhibitory factor (MIF) was first recognized as a lymphokine that prevents macrophage migration in vitro. However, subsequent studies have revealed that MIF also functions as a chemokine, recru...Human macrophage migration inhibitory factor (MIF) was first recognized as a lymphokine that prevents macrophage migration in vitro. However, subsequent studies have revealed that MIF also functions as a chemokine, recruiting monocytes, neutrophils, and lymphocytes to sites of inflammation. While the role of its homologue, D-dopachrome tautomerase (D-DT or MIF-2), is less well understood, it shares both unique and overlapping functions with MIF. Both MIF and D-DT exhibit tautomerase catalytic activity, with MIF's tautomerase active site being associated with health, but also the pathogenesis of various inflammatory conditions, autoimmune diseases, and cancers. In contrast, the role of D-DT's tautomerase active site in disease remains poorly understood. In this study, we present a method for evaluating the tautomerase activity and inhibition of recombinant purified MIF and D-DT, using 4-hydroxyphenylpyruvate (4-HPP) and phenylpyruvate (PP) as substrates. Although both substrates can be used to measure MIF tautomerase activity, 4-HPP is preferred, while PP is favored for D-DT, highlighting the distinct substrate specificities of the two enzymes.
Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine implicated in the pathogenesis of multiple acute and chronic inflammatory conditions, harbours diverse catalytic activity, notably the ability to...Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine implicated in the pathogenesis of multiple acute and chronic inflammatory conditions, harbours diverse catalytic activity, notably the ability to tautomerize 2-carboxy-2,3-dihydroxyindole-5,6-quinone (L-dopachrome) to its dihydroxyindole derivative. While an endogenous substrate for this tautomerase activity is yet to be fully characterized, it is possible MIF enzymatic activity is important for numerous biological functions. As a result, assessing the tautomerase activity of MIF may serve as a useful screening tool for identifying small-molecule MIF inhibitors. This chapter details the use of a simple, cell-free, spectrophotometric assay for investigating the inhibitory potential of small-molecule compounds against the keto-enol tautomerase activity of MIF.