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Staging of Mouse Seminiferous Tubule Cross-Sections

Spermatogenesis is a cyclic process during which, within each epithelial area, various generations of germ cells undergo a series of developmental steps according to a fixed time schedule. The cycle of the seminiferous epithelium can be subdivided into stages. In the mouse, 12 such stages ha ...

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Isolation and Short-Term Culture of Mouse Spermatocytes for Analysis of Meiosis

Understanding meiosis is facilitated by in vitro experimental approaches, but this has not been easily applicable to mammalian meiocytes. Available methods for in vitro analysis of mammalian oocytes are generally limited to experimental analysis of the late prophase period. Short ...

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Isolation and Analyses of Enriched Populations of Male Mouse Germ Cells by Sedimentation Velocity: The Centrifugal Elutriation

The studies of molecular events that occur in single cell types within a tissue often require the disaggregation of the tissue into a single cell suspension, followed by isolation of distinct cell populations. The germinal epithelium of mammals is composed of several cell types, which divide ...

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Analyzing Mammalian Female Meiosis

The goal of this chapter is twofold: First, to acquaint the reader with the problems inherent in analyzing mammalian female meiosis and, second, to provide a step-by-step approach to mastering the necessary techniques. Although the methods presented are for use in the human and mouse, with subtle ...

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Immunofluorescence Analysis of Human Spermatocytes

New immunofluorescence techniques allow visual identification of human cells in various stages of meiotic prophase. Antibodies to the synaptonemal complex, the centromere and sites of recombination allow these stages of meiotic prophase to be identified. The progress of chromo ...

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Using RNA FISH to Study Gene Expression During Mammalian Meiosis

During mouse meiosis, gene expression and homologous synapsis are intimately linked. Chromosomes that fail to synapse at the zygotene–pachytene transition become transcriptionally silenced by a process called Meiotic Silencing of Unsynapsed Chromatin (MSUC), and this sile ...

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Beads on the Run: Beads as Alternative Tools for Chemotaxis Assays

Cell migration is required for a wide variety of processes from bacteria seeking for food to correct patterning of neuronal networks. The ability to sense external cues is critical for cells to get directions and reach their goals. So far, studies on chemotaxis have mainly focused their attenti ...

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Quantification of Transendothelial Migration Using Three-Dimensional Confocal Microscopy

Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using c ...

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In Vitro Analysis of Chemotactic Leukocyte Migration in 3D Environments

Cell migration on two-dimensional (2D) substrates follows entirely different rules than cell migration in three-dimensional (3D) environments. This is especially relevant for leukocytes that are able to migrate in the absence of adhesion receptors within the confined geometry of ...

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Endothelial Cell Migration Under Flow

The endothelial cells lining blood vessels are continuously exposed to fluid shear stress generated by pulsatile flow of blood. In order to minimise forces acting on their surface, endothelial cells adapt to shear stress by alignment and migration within the direction of flow. Failure to ad ...

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Cell Migration in Confinement: A Micro-Channel-Based Assay

This chapter describes a method to study cells migrating in micro-channels, a confining environment of well-defined geometry. This assay is a complement to more complex 3D migration systems and provides several advantages even if it does not recapitulate the full complexity of 3D migrati ...

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Measuring FRET Using Time-Resolved FLIM

Cell migration is a process that is controlled by the formation and correct localization of protein complexes and by post-translational modification of individual proteins. Forster or fluorescent resonance energy transfer (FRET) detected using fluorescence lifetime imagi ...

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Fluorescence Recovery After Photobleaching

This chapter describes the use of microscope-based fluorescence recovery after photobleaching (FRAP). To quantify the dynamics of proteins within a subcellular compartment, we first outline the general aspects of FRAP experiments and then provide a detailed protocol of how to measu ...

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Functional Screening with a Live Cell Imaging-Based Random Cell Migration Assay

Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrins ...

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Characterizing System Performance in Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy (TIRF-M) has become an increasingly popular tool to study events in close proximity to the cell cortex, such as cell adhesion (Axelrod, J Cell Biol 89:141–145, 1981; Gingell et al., J Cell Biol 100:1334–1338, 1985; Patel et al., J Cell Sci 121:1159–11 ...

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Time-Lapse Imaging of Chick Cardiac Precursor Cells

The chick embryo is easily accessible and has therefore been widely used in developmental biology studies. In particular, the early embryo can be removed from the egg and cultured, which allows real-time observations and imaging. Here, we describe ex vivo electroporation followed by long- ...

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Measuring Angiogenesis in Mice

Angiogenesis is the formation of new blood vessels from pre-existing vessels. This process is essential during embryonic development, wound healing, and regeneration as well as during several pathological conditions such as cancer. In this chapter, we describe an assay to measure angi ...

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Two-Photon Intravital Multicolour Imaging to Study Metastatic Behaviour of Cancer Cells In Vivo

In the last decade, intravital microscopy on breast tumours in mice at single-cell resolution has resulted in important new insight into mechanisms of metastatic behaviour such as migration, invasion, and intravasation of tumour cells; angiogenesis; and the response of immune cells. T ...

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Simple Experimental and Spontaneous Metastasis Assays in Mice

Many steps of the metastatic cascade can be reproduced in simple in vitro assays such as tumour cell interactions with matrix proteins, proteolysis, chemotaxis, haptotaxis, and invasion into matrices or explanted tissues. Nevertheless, there are no fully adequate substitutes for the c ...

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Assessment of Development and Chemotaxis in Dictyostelium discoideum Mutants

Studies using the social amoeba Dictyostelium discoideum have greatly contributed to the current understanding of the signaling network that underlies chemotaxis. Since directed migration is essential for normal D. discoideum multicellular development, mutants with che ...

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