| 1. |
|
|
| 2. |
- Al-Jubair, Tamim, et al.
(author)
-
Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)
- 2022
-
In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
-
Journal article (peer-reviewed)abstract
- Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST). MST has the advantage of low sample consumption and high detergent compatibility enabling AQP protein-protein interaction investigation with a high level of control of components and environment. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Roche et al. (2017).
|
|
| 3. |
- Al-Jubair, Tamim, et al.
(author)
-
High-yield overproduction and purification of human aquaporins from Pichia pastoris
- 2022
-
In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
-
Journal article (peer-reviewed)abstract
- Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1–5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).
|
|
| 4. |
- Arellano-Caicedo, Carlos, et al.
(author)
-
Quantification of growth and nutrient consumption of bacterial and fungal cultures in microfluidic microhabitat models
- 2024
-
In: STAR Protocols. - 2666-1667. ; 5:1
-
Journal article (peer-reviewed)abstract
- Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023).1
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Bojmar, Linda, 1983-, et al.
(author)
-
Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids
- 2021
-
In: STAR protocols. - : Cell Press. - 2666-1667. ; 2:1
-
Journal article (peer-reviewed)abstract
- We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).
|
|
| 8. |
- Bojmar, Linda, et al.
(author)
-
Protocol for cross-platform characterization of human and murine extracellular vesicles and particles
- 2024
-
In: STAR PROTOCOLS. - : ELSEVIER. - 2666-1667. ; 5:1
-
Journal article (peer-reviewed)abstract
- Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross -validation across different platforms.For complete details on the use and execution of this protocol, please refer to Hoshino et al.1
|
|
| 9. |
|
|
| 10. |
- Boutet-Robinet, Elisa, et al.
(author)
-
Detection of DNA damage by alkaline comet assay in mouse colonic mucosa
- 2021
-
In: STAR Protocols. - : Cell Press. - 2666-1667. ; 2:4
-
Journal article (peer-reviewed)abstract
- We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).
|
|
| 11. |
- Bronnec, Vicky, et al.
(author)
-
Detailed protocol for germ-free Drosophila melanogaster colonization with Propionibacterium spp. biofilms
- 2022
-
In: STAR Protocols. - : Cell Press. - 2666-1667. ; 3:2
-
Journal article (peer-reviewed)abstract
- In this protocol, we describe a germ-free Drosophila melanogaster model to investigate anaerobic bacterial biofilms. We detail how to establish Propionibacterium spp. biofilms in the fruit fly's gut using an easy to carry out method. For complete details on the use and execution of this protocol, please refer to Bronnec and Alexeyev (2021) and Bronnec et al. (2022).
|
|
| 12. |
|
|
| 13. |
|
|
| 14. |
- Erttmann, Saskia F., et al.
(author)
-
Protocol for isolation of microbiota-derived membrane vesicles from mouse blood and colon
- 2023
-
In: STAR Protocols. - : CellPress. - 2666-1667. ; 4:1
-
Journal article (peer-reviewed)abstract
- Bacterial membrane vesicles have emerged as gadgets allowing remote communication between the microbiota and distal host organs. Here we describe a protocol for enriching vesicles from serum and colon that could widely be adapted for other tissues. We detail pre-clearing of serum or colon fluids using 0.2-μm syringe filters and their concentration by centrifugal filter devices. We also describe vesicle isolation with qEV size exclusion columns and finally the concentration of isolated vesicle fractions for downstream analyses. For complete details on the use and execution of this protocol, please refer to Erttmann et al. (2022).1
|
|
| 15. |
|
|
| 16. |
|
|
| 17. |
|
|
| 18. |
- Generó, Magalí Martí, 1983-, et al.
(author)
-
A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials
- 2021
-
In: STAR Protocols. - Cambridge, MA, United States : Cell press. - 2666-1667. ; 2:3
-
Journal article (peer-reviewed)abstract
- 16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic L. reuteri identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiotic supplementation and clinical outcomes. It is customizable for other clinical trials. For complete details on the use and execution of this protocol, please refer to Martí et al. (2021) and Spreckels et al. (2021).
|
|
| 19. |
- Giacomoni, Jessica, et al.
(author)
-
Protocol for optical clearing and imaging of fluorescently labeled ex vivo rat brain slices
- 2023
-
In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 4:1
-
Journal article (peer-reviewed)abstract
- Tissue clearing is commonly used for whole-brain imaging but seldom used for brain slices. Here, we present a simple protocol to slice, immunostain, and clear sections of adult rat brains for subsequent high-resolution confocal imaging. The protocol does not require toxic reagents or specialized equipment. We also provide instructions for culturing of rat brain slices free floating on permeable culture inserts, maintained in regular CO2 incubators, and handled only at media change.
|
|
| 20. |
|
|
| 21. |
- Ignatov, Dmitriy, et al.
(author)
-
Generation of Sequencing Libraries for Structural Analysis of Bacterial 5′ UTRs
- 2020
-
In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 1:2
-
Journal article (peer-reviewed)abstract
- The structure of 5′ untranslated regions (5′ UTRs) of bacterial mRNAs often determines the fate of the transcripts. Using a dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) approach, we developed a protocol to generate sequence libraries to determine the base-pairing status of adenines and cytosines in the 5′ UTRs of bacterial mRNAs. Our method increases the sequencing depth of the 5′ UTRs and allows detection of changes in their structures by sequencing libraries of moderate sizes. For complete details on the use and execution of this protocol, please refer to Ignatov et al. (2020).
|
|
| 22. |
|
|
| 23. |
- Johansson, Pia Annette, et al.
(author)
-
CRISPRi-mediated transcriptional silencing in iPSCs for the study of human brain development
- 2022
-
In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
-
Journal article (peer-reviewed)abstract
- This protocol describes the design and use of CRISPRi-mediated transcriptional silencing in human iPSCs, for loss-of-function studies in brain development research. The protocol avoids single cell selection, thereby eliminating side effects of clonal expansion and sites of viral integration. We also describe a neural progenitor differentiation protocol and discuss the challenges of target-specific lentiviral silencing, efficient silencing levels, and off-target effects. For complete details on the use and execution of this protocol, please refer to Johansson et al. (2022).
|
|
| 24. |
- Kawale, Ashish A., et al.
(author)
-
Characterization of backbone dynamics using solution NMR spectroscopy to discern the functional plasticity of structurally analogous proteins
- 2021
-
In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 2:4
-
Journal article (peer-reviewed)abstract
- The comprehensive delineation of inherent dynamic motions embedded in proteins, which can be crucial for their functional repertoire, is often essential yet remains poorly understood in the majority of cases. In this protocol, we outline detailed descriptions of the necessary steps for employing solution NMR spectroscopy for the in-depth amino acid level understanding of backbone dynamics of proteins. We describe the application of the protocol on the structurally analogous Tudor domains with disparate functionalities as a model system. For complete details on the use and execution of this protocol, please refer to Kawale and Burmann (2021). © 2021 The Author(s)
|
|
| 25. |
- Kitchen, Philip, et al.
(author)
-
Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells
- 2020
-
In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 1:3
-
Journal article (peer-reviewed)abstract
- Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).
|
|
