Current protocols最新文献

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood.

Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Whole blood aggregometry in mice

Support Protocol: Phenylhydrazine-induced anemia in wild-type mice

Basic Protocol 2: Hematocrit percentage in mice

Microplastics (MPs; 1 µm to 5 mm) are a persistent and pervasive environmental pollutant of emergent and increasing concern. Human exposure to MPs through food, water, and air has been documented and thus motivates the need for a better understanding of the biological implications of MP exposure. These impacts are dependent on the properties of MPs, including size, morphology, and chemistry, as well as the dose and route of exposure. This overview offers a perspective on the current methods used to assess the bioactivity of MPs. First, we discuss methods associated with MP bioactivity research with an emphasis on the variety of assays, exposure conditions, and reference MP particles that have been used. Next, we review the challenges presented by common instrumentation and laboratory materials, the lack of standardized reference materials, and the limited understanding of MP dosimetry. Finally, we propose solutions that can help increase the applicability and impact of future studies while reducing redundancy in the field. The excellent protocols published in this issue are intended to contribute toward standardizing the field so that the MP knowledge base grows from a reliable foundation. © 2024 Wiley Periodicals LLC.

The intestinal inflammation induced by injection of naïve CD4⁺ T cells into lymphocyte-deficient hosts (more commonly known as the T cell transfer model of colitis) shares many features of idiopathic inflammatory bowel disease (IBD) in humans, such as epithelial cell hyperplasia, crypt abscess formation, and dense lamina propria lymphocyte infiltration. As such, it provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. In the IBD model described here, colitis is induced in Rag (recombination-activating gene)-deficient mice by reconstitution of these mice with naïve CD4⁺CD45RB^hi T cells through adoptive T cell transfer. Although different recipient hosts of cell transfer can be used, Rag-deficient mice are the best characterized and support studies that are both flexible and reproduceable. As described in the Basic Protocol, in most studies the transferred cells consist of naïve CD4⁺ T cells (CD45RB^hi T cells) derived by fluorescence-activated cell sorting from total CD4⁺ T cells previously purified using immunomagnetic negative selection beads. In a Support Protocol, methods to characterize colonic disease progression are described, including the monitoring of weight loss and diarrhea and the histological assessment of colon pathology. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Induction of IBD in Rag-deficient mice by the transfer of naïve CD4⁺CD45RB^hi T cells

Support Protocol: Monitoring development of colitis

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs)

Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels.

Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture.

Support Protocol 2: Organoid fixation with paraformaldehyde (PFA)

Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids.

Basic Protocol 3: Immunofluorescence of organoid cryosections

Current Protocols is issuing corrections for the following protocol article.

Kannan, S. K., Kim, C. Y., Heidarian, M., Berton, R. R., Jensen, I. J., Griffith, T. S., & Badovinac, V. P. (2024). Mouse models of sepsis. Current Protocols, 4, e997. doi: 10.1002/cpz1.997

In the above-referenced article:

A sentence has been added to the Acknowledgements to thank Dr. Patricia De Assis for her contribution to this article.

The current version online now includes these corrections and may be considered the authoritative version of record.

Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Enrichment of mucin-domain glycoproteins

Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s)

Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides

Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides

Cardiovascular diseases have emerged as one of the leading causes of human mortality, but the discovery of new drugs has been hindered by the absence of suitable in vitro platforms. In recent decades, continuously refined protocols for differentiating human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs) have significantly advanced disease modeling and drug screening; however, this has led to an increasing need to monitor the function of hiPSC-CMs. The precise regulation of action potentials (APs) and intracellular calcium (Ca²⁺) transients is critical for proper excitation-contraction coupling and cardiomyocyte function. These important parameters are usually adversely affected in cardiovascular diseases or under cardiotoxic conditions and can be measured using optical imaging–based techniques. However, this procedure is complex and technologically challenging. We have adapted the IonOptix system to simultaneously measure APs and Ca²⁺ transients in hiPSC-CMs loaded with the fluorescent dyes FluoVolt and Rhod 2, respectively. This system serves as a powerful high-throughput platform to facilitate the discovery of new compounds to treat cardiovascular diseases with the cellular phenotypes of abnormal APs and Ca²⁺ handling. Here, we present a comprehensive protocol for hiPSC-CM preparation, device setup, optical imaging, and data analysis. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Maintenance and seeding of hiPSC-CMs

Basic Protocol 2: Simultaneous detection of action potentials and Ca²⁺ transients in hiPSC-CMs

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Identify the impact of a metal on the competence of HSCs

Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation

Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure

Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs

Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure

Current Protocols is issuing corrections for the following protocol article.

Bryant, J. D., Lei, Y., VanPortfliet, J. J., Winters, A. D., & West, A. P. (2022). Assessing mitochondrial DNA release into the cytosol and subsequent activation of innate immune-related pathways in mammalian cells. Current Protocols, 2, e372. doi: 10.1002/cpz1.372

In the above-referenced article:

In Basic Protocol 2, step 13, the tube label has been changed from “B-2” to “A-2”.

In Basic Protocol 2, the following instruction has been added to step 16: “Save the pellet to use in step 21.”

In Basic Protocol 2, step 21, the step number has been changed from “step 18” to “step 16”.

In Basic Protocol 2, the following instruction has been added to step 23: “Save the pellet to use in step 26.”

The current version online now includes these corrections and may be considered the authoritative version of record.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining

Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System

Basic Protocol 3: Manual De Novo Assembly workflow

Basic Protocol 4: Local guided assembly workflow

Basic Protocol 5: EnFocus Fragile X workflow

Basic Protocol 6: Molecule distance script workflow