Editor
Andrea Puhar
  • Faculty, The Wellcome-Wolfson Institute for Experimental Medicine (WWIEM), Queen's University Belfast
Research fields
  • Immunology, Microbiology
Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting
To cause an infection, the human specific pathogen Neisseria meningitides must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, N. meningitidis may cross the epithelial cellular barrier, reach the bloodstream and cause sepsis and/or meningitis. Since N. meningitidis niche is restricted to humans the availability of relevant animal models to study host-pathogen interactions are limiting. Therefore, most findings that involve N. meningitidis colonization derive from studies using cultured human cell lines. Human epithelial cells have been successfully used to examine and identify molecular effectors involved in initial adherence of the pathogen. Here, we describe a standard protocol to quantify the adherence of N. meningitidis to epithelial pharyngeal FaDu cells. Colony counts of cell lysates collected after infection are used to quantify adherence to the epithelial cells.
Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 12073, Q&A: 1
In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter]

To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources.

Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users’ need.

Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al., 2017). We report here only the generation of the delivery vehicle i.e., the fabrication of Cas9En-RNP/ArgNPs nanoassembly.
Ubiquitination Assay for Mammalian Cells
Authors:  Yang Peng, Edward Wang, Guang Peng and Shiaw-Yih Lin, date: 07/20/2016, view: 23531, Q&A: 2
Ubiquitin is an 8.5 kDa protein that can be activated and conjugated by ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, respectively. Ubiquitin E3 ligases then recognize protein substrates, and then transfer the ubiquitin from E2 to the targeted protein. This biological process is called ubiquitination, and it is an important biological process which can signal protein degradation via the proteasome. The aim of this protocol is to describe a procedure that determines the level of cellular ubiquitination in a protein-of-interest relative to control cells.
Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet
Authors:  Kristofor K. Ellestad and Colin C. Anderson, date: 01/05/2016, view: 31543, Q&A: 0
Measurement of the incorporation of radionuclides such as 3H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately quantitate T cell divisions, and an inability to combine proliferation analyses with simultaneous evaluation of the expression of cellular markers in divided cells. By labeling T cells with reactive dyes such as CFSE, Celltrace Violet, and others that are partitioned equally between daughter cells at each cell division, one can relatively easily track generations of proliferated cells and their expression of various molecules by flow cytometry.

FoxP3+ regulatory T cells (Treg) are critical mediators of immune tolerance and evaluation of their functionality is an important step in characterizing many immune models (Rudensky, 2011). Classically CD4+ Treg and conventional or “responder” T cells have been isolated based on their surface expression of CD25 (Treg: CD4+CD25+, Tresp: CD4+CD25-). However, we and others have noted that populations of CD4+CD25- cells express the FoxP3 transcription factor and have suppressive function. Therefore we have utilized the transgenic FoxP3-EGFP mouse to facilitate live purification of suppressor and responder populations based on EGFP (and thus FoxP3) expression. Here we present our adapted protocol for assaying regulatory T cell suppression of Celltrace Violet-labeled responder T cells.
Two-event Transfusion-related Acute Lung Injury Mouse Model
Authors:  Guadalupe Ortiz-Muñoz and Mark R. Looney, date: 06/20/2015, view: 8840, Q&A: 0
Transfusion-related acute lung injury (TRALI) is defined as acute lung injury that occurs within 6 hours of a blood product transfusion. TRALI continues to be a leading cause of transfusion-related mortality and we have developed a mouse model of TRALI to better understand the mechanisms by which injury occurs and to test therapeutic approaches. Our model is a two-event model based on immune priming and the challenge of BALB/c wild-type mice with cognate MHC Class I monoclonal antibody (MHC I mAb). Immune priming with LPS mimics the primed state of recipients (first event) that is important for the development of TRALI. Donor HLA antibodies are frequently implicated in TRALI reactions, and cognate MHC Class I antibody (second event) produces acute lung injury in primed animals. Here, we describe a detailed protocol with high reproducibility within animals.
Non-invasive Intratracheal Instillation in Mice
Authors:  Guadalupe Ortiz-Muñoz and Mark R. Looney, date: 06/20/2015, view: 18843, Q&A: 1
The intratracheal instillation technique is used to deliver a variety of agents to the lungs ranging from pathogens (bacteria, viruses), toxins, to therapeutic agents. To model lung inflammation and injury, LPS can be administrated via intranasal, intratracheal, or aerosol approaches. Each technique has its limitations. The intratracheal technique can involve the non-invasive instillation method (via the oro-tracheal route) or a direct injection into the trachea. Here, we describe an optimized method for direct visual instillation of LPS via the non-invasive oro-tracheal route.
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