| 1. |
- Bag, Pushan, 1993-, et al.
(author)
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Solubilization method for isolation of photosynthetic mega- And super-complexes from conifer thylakoids
- 2021
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In: Bio-protocol. - 2331-8325. ; 11:17
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Journal article (peer-reviewed)abstract
- Photosynthesis is the main process by which sunlight is harvested and converted into chemical energy and has been a focal point of fundamental research in plant biology for decades. In higher plants, the process takes place in the thylakoid membranes where the two photosystems (PSI and PSII) are located. In the past few decades, the evolution of biophysical and biochemical techniques allowed detailed studies of the thylakoid organization and the interaction between protein complexes and cofactors. These studies have mainly focused on model plants, such as Arabidopsis, pea, spinach, and tobacco, which are grown in climate chambers even though significant differences between indoor and outdoor growth conditions are present. In this manuscript, we present a new mild-solubilization procedure for use with “fragile” samples such as thylakoids from conifers growing outdoors. Here, the solubilization protocol is optimized with two detergents in two species, namely Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). We have optimized the isolation and characterization of PSI and PSII multimeric mega- and super-complexes in a close-to-native condition by Blue-Native gel electrophoresis. Eventually, our protocol will not only help in the characterization of photosynthetic complexes from conifers but also in understanding winter adaptation.
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| 2. |
- Håglin, Sofia, et al.
(author)
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Single or Repeated Ablation of Mouse Olfactory Epithelium by Methimazole
- 2021
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In: Bio-protocol. - : Bio-protocol. - 2331-8325. ; 11:8
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Journal article (peer-reviewed)abstract
- Odor-detecting olfactory sensory neurons residing in the nasal olfactory epithelium (OE) are the only neurons in direct contact with the external environment. As a result, these neurons are subjected to chemical, physical, and infectious insults, which may be the underlying reason why neurogenesis occurs in the OE of adult mammals. This feature makes the OE a useful model for studying neurogenesis and neuronal differentiation, with the possibility for systemic as well as local administration of various compounds and infectious agents that may interfere with these cellular processes. Several different chemical compounds have been shown to cause toxic injury to the OE, which can be used for OE ablation. We, and others, have found that the systemic administration of the hyperthyroid drug, methimazole, reliably causes olfactotoxicity as a side effect. Here, we outline an OE lesioning protocol for single or repeated ablation by methimazole. A single methimazole administration can be used to study neuroepithelial regeneration and stem cell activation, while repeated ablation and regeneration of OE enable the study of tissue stem cell exhaustion and generation of tissue metaplasia.
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| 3. |
- Jakic, Bojana, et al.
(author)
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Bacterial Infection with Listeria monocytogenes in Mice and Subsequent Analysis of Antigen-Specific CD8 T Cell Responses
- 2021
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In: Bio-protocol. - : BIO-PROTOCOL. - 2331-8325. ; 11:23
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Journal article (peer-reviewed)abstract
- Pathogens such as bacteria, viruses, fungi, or protozoa can cause acute and chronic infections in their hosts. The intracellular bacterium Listeria monocytogenes serves as a model pathogen to assess the molecular mechanisms regulating CD8 T cell activation, differentiation, and function. We set up an experimental workflow to investigate cell-intrinsic roles of the nuclear receptor NR2F6 in CD8 T cell memory formation upon Listeria monocytogenes (LmOVA) infection (Jakic et al., 2021). The current protocol details how to cultivate ovalbum in-expressing LmOVA, infect naive C57BL/6 mice with these bacteria and determine the bacterial load in host organs. Furthermore, we describe how to evaluate antigen-specific CD8 T cell responses and discriminate between short-lived effector and memory precursor cells in vivo following LmOVA infection (Figure 1). To assess CD8 T cell-intrinsic molecular mechanisms, we integrated an adoptive cell transfer (ACT) experiment of genetically modified naive OT-I CD8 T cells into congenic hosts before LmOVA infection. [GRAPHICS] .
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| 4. |
- Pastula, Agnieszka, et al.
(author)
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Induction of Epithelial-mesenchymal Transition in MDCK II Cells
- 2021
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In: Bio-protocol. - : Bio-protocol. - 2331-8325. ; 11:3
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Journal article (peer-reviewed)abstract
- Epithelial-mesenchymal transition (EMT) is a reversible process of epithelial cell transdifferentiation into a mesenchymal cell, that enables initiation of cell migration. EMT plays an important role in embryonic development, tissue repair and cancer metastasis. Better understanding of cellular and molecular events during EMT will not only provide novel insights on how mammalian organism develops and how epithelial tissues regenerate, but also can identify novel therapeutic targets for cancer therapy. Here we aim to provide a detailed protocol on how to induce EMT in Madin-Darby Canine Kidney (MDCK) II epithelial cell line and perform immunofluorescent staining on EMT-induced cells.
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| 5. |
- Rocha, A. L., et al.
(author)
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A Method to Induce Brown/Beige Adipocyte Differentiation from Murine Preadipocytes
- 2021
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In: Bio-Protocol. - 2331-8325. ; 11:24
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Journal article (peer-reviewed)abstract
- Adipocytes exhibit different morphological and functional characteristics, depending on their anatomical location, developmental origin, and stimulus. While white adipocytes tend to accumulate energy as triglycerides, brown and beige adipocytes tend to direct carbon sources to fuel thermogenesis. White and beige adipocytes originate from common progenitor cells, which are distinct from brown adipocyte precursors. Having a method to study white vs. beige vs. brown adipocyte differentiation may help to unveil the mechanisms driving distinct adipogenic programs. Preadipocytes can be cultured and differentiated in vitro using a combination of compounds to stimulate adipogenesis. Here, we describe and compare protocols designed to stimulate adipocyte differentiation and induce brown/beige-like or white-like characteristics in differentiating adipocytes. The protocols consist in exposing murine preadipocytes to pharmacological stimuli aimed at triggering adipogenesis and inducing (or not) a thermogenic gene expression program. After 8 days of differentiation with a pro-browning cocktail, immortalized preadipocytes isolated from interscapular brown fat (9B cells) or inguinal white fat (9W cells) from the same mouse expressed higher levels of brown/beige adipocyte markers (e.g., Ucp1) and pan-adipocyte differentiation markers (e.g., Pparg, Cebpa and aP2) when compared to the same cells differentiated with a cocktail that lacked brown/beige adipogenic inducers (i.e., rosiglitazone, T3, and indomethacin). Consistent with a higher thermogenic potential of brown vs. beige adipocytes, differentiated 9B cells expressed higher Ucp1 levels than differentiated 9W cells. This simple protocol may help researchers to understand mechanisms of adipogenesis and how adipocytes become thermogenic.
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| 6. |
- Shilling, Patrick J., et al.
(author)
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Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production
- 2021
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In: Bio-protocol. - 2331-8325. ; 11:16
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Journal article (peer-reviewed)abstract
- pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used modern methods of DNA assembly and directed evolution to identify improved designs for these modules and demonstrated that these designs support higher protein production yields. Herein, we present two PCR protocols for implementing the designs and increasing protein production from existing pET expression plasmids.
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