Flow cytometry is the method of choice to ‘diagnose’ paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used.
The lipophilic cation JC‐1 (5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨm).
Determination of incorporation of the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle.
Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized.
Using flow cytometry, single‐cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics.
Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume of adherent cells using a standard light microscope.
Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck.
FISH has been used to detect and clarify deletions and/or other structural rearrangements, and also has applications in interphase analysis. This unit describes preparation of uncultured amniotic fluid cells for FISH analysis.
Diagnosing constitutional pathogenic copy number variants (CNVs) requires detecting submicroscopic segmental chromosomal imbalances. The Affymetrix GeneChip mapping array was one of the initial microarray platforms used to measure duplication and deletion of genetic material in DNA samples.
Most applications for RNA‐seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits.
The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials.
Proteasome inhibitors are indispensable research tools in immunology and cell biology. With numerous proteasome inhibitors available commercially, choosing the appropriate compound for a biological experiment may be challenging, especially for a novice.
This unit presents assays that allow accurate measurement of phagocytosis and killing of bacteria by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria.
The mammalian Toll‐like receptor (TLR) family consists of 13 members, and recognizes specific patterns of microbial components, called pathogen‐associated molecular patterns (PAMPs).
Human membranous nephritis is a major cause of end‐stage kidney disease. Active Heymann nephritis (HN) is an auto‐immune model of membranous nephritis induced in Lewis rats by immunization with a crude renal tubular antigen (Fx1A) or megalin (gp330).
The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full‐length cDNAs in species‐specific expression vectors and subsequent functional analysis of the expressed protein.
Most eukaryotic genes are transcribed into mRNAs with alternative poly(A) sites. Emerging evidence suggests that mRNA isoforms with alternative poly(A) sites can perform critical regulatory functions in numerous biological processes.
Cultured mammalian cells provide an environment ideal for producing functional recombinant mammalian proteins. However, low expression levels of recombinant proteins present a challenge for their detection and purification.
Protein, peptides, and nucleic acids are biomolecules that drive biological processes in living organisms.
Occupied Regions of Genomes from Affinity‐purified Naturally Isolated Chromatin (ORGANIC) is a high‐resolution method that can be used to quantitatively map protein‐DNA interactions with high specificity and sensitivity.
Acute brain slices are widely used in neuroscience because this preparation enables pharmacological interventions in a timely manner, similar to what is currently done in cultured cell studies, while preserving the natural cytoarchitecture.
Neuronal migration is one of the fundamental processes underlying the proper assembly and function of neural circuitry. The majority of neuronal precursors are generated far away from their sites of integration and need to migrate substantial distances to reach their final destination.
Latent sensitization is a rodent model of chronic pain that reproduces both its episodic nature and its sensitivity to stress. It is triggered by a wide variety of injuries ranging from injection of inflammatory agents to nerve damage.
When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science.
PDZ domains function in nature as protein‐binding domains within scaffold and membrane‐associated proteins. They comprise approximately 90 residues and undergo specific, high‐affinity interactions with complementary C‐terminal peptide sequences, other PDZ domains, and/or phospholipids.
Tyrosine sulfation is a post‐translational modification (PTM) where a sulfate group is added to a tyrosine moiety. This PTM is responsible for strengthening interaction between proteins.
This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments.
This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids.