A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non-Vesicular Organelles.

A wide range of fluorescent dyes and reagents exist for labeling organelles in live and fixed cells. Choosing between them can lead to confusion, and optimization for many of them can be challenging. Presented here is a discussion on the commercially available reagents that have shown the most promise for each organelle of interest, including endoplasmic reticulum/nuclear membrane, Golgi apparatus, mitochondria, nucleoli, and nuclei, with an emphasis on localization of these structures for microscopy. Included is a featured reagent for each structure with a recommended protocol, troubleshooting guide, and example image. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Endoplasmic reticulum and nuclear membrane labeling using ER-Tracker reagents Basic Protocol 2: Labeling Golgi apparatus using dye-labeled ceramides Basic Protocol 3: Labeling mitochondria using MitoTracker Red CMXRos Basic Protocol 4: Labeling nucleoli using SYTO RNASelect Green.

A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part I: BacMam Labeling and Reagents for Vesicular Structures.

Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has provided more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent, recommended protocol, troubleshooting guide, and example image for each structure. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Delivering targeted fluorescent proteins using pre-made, high-titer BacMam constructs Alternate Protocol 1: Non-pseudo-typed BacMam viruses in standard cell types and pseudo-typed BacMam viruses in hard-to-transduce cell types Basic Protocol 2: Labeling endosomes: pHrodo™-10k-dextran Basic Protocol 3: Labeling peroxisomes: BacMam 2.0 CellLight™ Peroxisome-GFP Alternate Protocol 2: Labeling peroxisomes using antibodies Basic Protocol 4: Labeling autophagosomes: Transduction of cells with Premo™ Autophagy Sensor GFP-LC3B Alternate Protocol 3: Labeling autophagosomes using antibodies Basic Protocol 5: Labeling lysosomes: LysoTracker Red DND-99.

A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part III: Reagents for Actin, Tubulin, Cellular Membranes, and Whole Cell and Cytoplasm.

Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to labeling tubulin and actin, with the key factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exists for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for labeling these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent, recommended protocol, troubleshooting guide, and example image for each structure. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Actin labeling Basic Protocol 2: Wheat germ agglutinin conjugates for plasma membrane labeling Basic Protocol 3: Labeling tubulin microtubules with Tubulin Tracker Deep Red Basic Protocol 4: Labeling whole cells or cytoplasm with 5(6)-CFDA SE.

Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.

Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. In the past, analysis of global protein phosphorylation has been accomplished through radiolabelling of samples with inorganic (32P or [gamma-32)P] ATP. The approach is limited to specimens amenable to radiolabelling and poses certain safety and disposal problems. Alternatively, immunodetection with antibodies to the common phosphoamino acids may be employed, but the antibodies are relatively expensive and exhibit limited specificity and a certain degree of cross-reactivity. Pro-Q Diamond dye is a new fluorescent phosphosensor technology suitable for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins directly in isoelectric focusing gels, SDS-polyacrylamide gels and two-dimensional gels. Additionally, the technology is appropriate for the detection of phosphoproteins or phosphopeptides arrayed on protein chips or affixed to beads. Dye-stained proteins and peptides can be excited with a laser-based light source of 532 or 543 nm or with a xenon-arc lamp-based system equipped with appropriate band pass filters. Alternatively, ultraviolet light of about 302 nm may be employed, providing that sufficiently long exposure times are used to collect the fluorescence signal. Pro-Q Diamond dye emits maximally at approximately 580 nm. The fluorescence-based detection technology is easy to conduct, cost effective and allows rapid large-scale screening of protein and peptide phosphorylation in a variety of solid-phase assay formats.

A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.

Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image.

A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures.

Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent for each structure, recommended protocol, troubleshooting guide, and example image.

A review of reagents for fluorescence microscopy of cellular compartments and structures, Part II: reagents for non-vesicular organelles.

A wide range of fluorescent dyes and reagents exist for labeling organelles in live and fixed cells. Choosing between them can sometimes be confusing, and optimization for many of them can be challenging. Presented here is a discussion on the commercially-available reagents that have shown the most promise for each organelle of interest, including endoplasmic reticulum/nuclear membrane, Golgi apparatus, mitochondria, nucleoli, and nuclei, with an emphasis on localization of these structures for microscopy. Included is a featured reagent for each structure with a recommended protocol, troubleshooting guide, and example image.