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Past Issue in 2019
Volume: 9, Issue: 9
Biochemistry
Separation of Natural Collagen Crosslinks Using Buffer and Ion-pairing Agent Free Solvents on Silica Hydride Column for Mass Spectrometry Detection
In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation exchange chromatography method has limited sensitivity and long run times while reverse phase chromatography requires strong ion-pairing. In this method, the sample containing crosslinks is applied on a diamond hydride column using water and acetonitrile solvents containing 0.1% (w/v) formic acid. Eight crosslinks are eluted separately from the column and detected by mass spectrometry in the sub-pmol range. By using this method, it is possible to separate all crosslinks of collagen in several biological samples without the need for ion-pairing agent or derivatization for detection.
Optimized Oxidative Stress Protocols for Low-microliter Volumes of Mammalian Plasma
Small blood volumes commonly obtained from small mammals during field studies are only sufficient for a single biochemical assay. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and developed modified methods to improve analytical selectivity and sensitivity required for measuring markers of oxidative stress using small blood volumes. Specifically, we proposed a modified malondialdehyde (MDA) analysis protocol by high performance liquid chromatography (HPLC) and also optimized both the uric acid independent ferric reducing antioxidant power (FRAP) and hypochlorous acid shock capacity (HASC) assays. We present methods in which a total volume of less than 60 μl of plasma is required to obtain a comprehensive portrait of an individual’s oxidative profile.
Cancer Biology
3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts with Human Prostate Cancer Epithelial Cells
Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression. This was established using transgenic mouse models. Paracrine interactions between fibroblasts and epithelial cells were further interrogated using isolated 2D cell culture systems, but 3D culture systems currently being developed can better mimic reciprocal interactions potentially found in the native tissue. To understand paracrine and juxtacrine signaling among fibroblasts and epithelia, 3D co-cultures with species differences allows for further subsequent analysis of the cultures. The use of mouse and human cells, for example, in one system allows for species-specific FACS or quantitative PCR analysis. This protocol describes the use of a 3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts with Human Prostate Cancer Epithelial Cells.
Cell Biology
Triple Fluorescence Anisotropy Reporter Imaging in Living Cells
FRET-based genetically encoded biosensors incorporate two fluorescent proteins into their design to enable ratiometric biosensing of signaling activities in live cells. While emission ratios are generally useful for quantitative studies, they leave little room in the optical spectrum for additional sensors and optogenetic tools. Homotransfer-based reporters, such as the FLuorescence Anisotropy REporters (FLAREs), incorporate two fluorescent proteins of the same color into their design. Conversion to a single color opens the visible spectrum for the use of complementary sensors. Here, we present a protocol for measuring three independent intracellular signals in living cells. We describe the configuration and calibration of a widefield microscope for multicolor FLARE imaging. Three FLARE sensors for intracellular calcium, MAPK activity, and PKA phosphorylation are co-transfected into HEK293 cells, and triple FRET imaging is performed. Compared to heterotransfer FRET biosensors, the polarization-based multiplex imaging can track multiple signaling activities concurrently in a targeted cell population.FRET-based genetically encoded biosensors incorporate two fluorescent proteins into their design to enable ratiometric biosensing of signaling activities in live cells. While emission ratios are generally useful for quantitative studies, they leave little room in the optical spectrum for additional sensors and optogenetic tools. Homotransfer-based reporters, such as the FLuorescence Anisotropy REporters (FLAREs), incorporate two fluorescent proteins of the same color into their design. Conversion to a single color opens the visible spectrum for the use of complementary sensors. Here, we present a protocol for measuring three independent intracellular signals in living cells. We describe the configuration and calibration of a widefield microscope for multicolor FLARE imaging. Three FLARE sensors for intracellular calcium, MAPK activity, and PKA phosphorylation are co-transfected into HEK293 cells, and triple FRET imaging is performed. Compared to heterotransfer FRET biosensors, the polarization-based multiplex imaging can track multiple signaling activities concurrently in a targeted cell population.
Quantification of Cutaneous Ionocytes in Small Aquatic Organisms
Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and environmental factors. Ionocytes profusely express ATPase enzymes, most notably Na+/K+ ATPase, which can be identified by immunohistochemistry. However, quantification of cutaneous ionocytes is not trivial due to the limited camera’s focal plane and the microscope’s field-of-view. This protocol describes a technique to consistently and reliably identify, image, and measure the relative surface area covered by cutaneous ionocytes through software-mediated focus-stacking and photo-stitching–thereby allowing the quantification of cutaneous ionocyte area as a proxy for ion transporting capacity across the skin. Because ionocytes are essential for regulating ionic composition, osmolarity, and acid/base status of internal fluids, this technique is useful for studying physiological mechanisms used by fish larvae and other small aquatic organisms during development and in response to environmental stress.
Microbiology
Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method
Many rhizobacteria isolated from plant rhizosphere produce various phytohormones in the form of secondary metabolites, the most common of which is Indole-3-acetic acid (IAA). Here, we detail analytical protocols of IAA detection and quantification, in vitro and in situ, as recently applied to Klebsiella SGM 81, a rhizobacterium isolated from the rhizosphere of Dianthus caryophyllus (a commercially important flower across the globe). Specifically, we describe a detailed protocol for a colorimetric assay using the Salkowski reagent method, which can be used to screen for the presence of Indole compounds. To further detect and quantify IAA, a highly accurate analytical approach of LC-MS/MS is used. To detect the presence of IAA around the root system of Dianthus caryophyllus, in situ staining of plant roots is done using Salkowski reagent.
Molecular Biology
Improved HTGTS for CRISPR/Cas9 Off-target Detection
Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a “bait” DNA double-stranded break (DSB) to capture genome-wide “prey” DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.
Neuroscience
Inducing Alcohol Dependence in Rats Using Chronic Intermittent Exposure to Alcohol Vapor
Alcohol use disorder (AUD) is a significant public health and economic burden and is often characterized by repeated bouts of alcohol intoxication and withdrawal. Virtually all organ systems are impacted by chronic alcohol exposure. These effects can be investigated using the rat as a model organism; however, rats typically will not self-administer alcohol to levels necessary to achieve physiological and behavioral aspects of dependence. The protocol described herein can be utilized to induce alcohol dependence in rats by administering alcohol vapor to the homecage for an extended period of time. This method allows the researcher to control the level, duration, and pattern of intoxication, and it reliably induces physiological and behavioral aspects of alcohol dependence, allowing for the study of biology and behavior with relevance for AUD in humans.
Plant Science
Determination of Root Exudate Concentration in the Rhizosphere Using 13C Labeling
One of the most remarkable metabolic features of plant roots is their ability to secrete a wide range of compounds into the rhizosphere, defined as the volume of soil around living roots. Around 5%-21% of total photosynthetically fixed carbon is transferred into the rhizosphere through root exudates. Until recently, studies on the quantity and quality of root exudates were conducted mostly under axenic or monoxenic in vitro conditions. Today, in situ assays are required to provide a better understanding of root exudates dynamics and role in plant-microbe interactions. By incubating plants with 13CO2 in situ for one week and quantifying 13C enrichment from the root-adhering soil using mass spectrometry, we were able to determine root exudate levels. Indeed, labeled substrate 13CO2 is converted into organic carbon via plant photosynthesis and transferred into the soil through root exudation. We assume that all 13C increases above natural abundance are mainly derived from exudates produced by 13C-labeled plants.
