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Molecular biology technique Tumor formation Tumor microenvironment Genome instability & mutation Inflammation Invasion & metastasis Microenvironment Oncogenesis Proliferative signaling Replicative immortality Tumor immunologyA Fast and Reliable Method to Generate Pure, Single Cell-derived Clones of Mammalian Cells
Stable cell cloning is an essential aspect of biological research. All advanced genome editing tools rely heavily on stable, pure, single cell-derived clones of genetically engineered cells. For years, researchers have depended on single-cell dilutions seeded in 96- or 192-well plates, followed by microscopic exclusion of the wells seeded with more than or without a cell. This method is not just laborious, time-consuming, and uneconomical but also liable to unintentional error in identifying the wells seeded with a single cell. All these disadvantages may increase the time needed to generate a stable clone. Here, we report an easy-to-follow and straightforward method to conveniently create pure, stable clones in less than half the time traditionally required. Our approach utilizes cloning cylinders with non-toxic tissue-tek gel, commonly used for immobilizing tissues for sectioning, followed by trypsinization and screening of the genome-edited clones. Our approach uses minimal cell handling steps, thus decreasing the time invested in generating the pure clones effortlessly and economically.
Graphical abstract:
A schematic comparison showing the traditional dilution cloning and the method described here. Here, a well-separated colony (in the green box) must be preferred over the colonies not well separated (in the red box).
Soft Agar Colony Formation Assay as a Hallmark of Carcinogenesis
Soft agar colony formation assay is established to estimate the anchorage-independent growth ability of cells. In this assay, a bottom layer of agar with complete media is poured and solidified first, followed by an upper layer containing a specified number of cells suspended in medium-agar mixture. After two weeks of incubation, the number of colonies will be counted, serving as an indicator of malignancy of tumor cells.
Isolation of Murine Alveolar Type II Epithelial Cells
We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. The purified alveolar type II epithelial cells can be cultured and maintained on fibronectin-coated plates in DMEM with 10% FBS. This protocol enables specific investigation of alveolar type II epithelial cells at molecular and cellular levels and provides an important tool to investigate in vitro the mechanisms underlying lung pathogenesis.
Murine Bronchoalveolar Lavage
A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, pathogens, proteins such as cytokines/chemokines, and the expression levels of inflammation-related and other genes in the cells. This will help to understand the underlying mechanisms of these lung diseases and develop specific and effective drugs.
Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen
Glioblastoma multiforme (GBM) is a grade 4 astrocytoma tumor in central nervous system. Astrocytes can be isolated from human GBM. Study of astrocytes can provide insights about the formation, progression and recurrence of glioblastoma. For using isolated astrocytes, new studies can be designed in the fields of pharmacology, neuroscience and neurosurgery for glioblastoma treatment. This protocol describes the details for preparing high purity primary astrocytes from human GBM. Tumor tissue is disrupted using mechanical dissociation and chemical digestion in this protocol. 2 weeks after plating the cell suspension in culture, primary astrocytes are available for further subculturing and immunocytochemistry of S100-beta antigen.
Generation of Mouse Thyroid Calcitonin-producing Cell Tumors from Primary Mouse Tumors
Medullary thyroid cancers (MTCs) are derived from calcitonin-producing cells (C cells) of neuroendocrine origin. Rb heterozygous mice develop low-grade C cell adenocarcinoma following biallelic inactivation of the Rb tumor suppressor gene loci. Additional inactivation of another tumor suppressor gene such as Trp53, Arf or Cdkn1a allows Rb-deficient mice to generate more aggressive C cell adenocarcinoma (Takahashi et al., 2006; Shamma et al., 2009; Kitajima et al., 2015). To characterize C cell adenocarcinoma cells derived from Rb-deficient mice of different genetic backgrounds, we attempted to extract C cell adenocarcinoma cells from primary thyroid tumor tissue. Since primary mouse small cell lung cancer (SCLC) cells those originate in neuroendocrine cells that also stems C cells, can be established both as non-adhesive and adhesive cells (Calbo et al., 2011), we applied their method to MTCs. Here we describe our isolation technique for non-adhesive and adhesive cell cultures from primary medullary thyroid tumor tissue. We found that the molecular markers of C cell such as Calcitonin and Ascl1 are predominantly enriched in the non-adhesive population (Kitajima et al., 2015). This is in line with the fact that one of most commonly distributed human MTC cell line TT is non-adhesive.
Analysis of Tumor-infiltrating Lymphocytes Following CD45 Enrichment
Measuring antigen-specific T cell responses in the blood and lymphoid organs of vaccinated mice can give us a useful indication of the potency of a vaccine formulation. Unfortunately, systemic or even localized lymphoid T cell responses are not always predictive of the ability of a vaccine to induce tumor protection. Measuring the antigen-specific T cell response within the tumor infiltrating lymphocytes is a more accurate indicator or vaccine efficacy. However, multi-parameter flow cytometric analysis of T cells isolated from tumor tissue can be quite challenging due to the over-whelming number of tumor cells present in relation to the tumor infiltrating lymphocytes (TIL) and to problems associated to the large and adhesive nature of many tumor cell. Here we take advantage of a pre-flow separation of CD45+ leukocytes from the tumor tissue using the MACS magnetic cell sorting system, resulting in a much cleaner cell preparation with which to proceed to flow cytometric staining and analysis.
Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells
Bone is a primary site of metastasis from prostate and breast cancers. Bone marrow macrophages (BMMs) are mediators of inflammatory processes and are thought to promote tumor growth in the skeletal sites. In order to elucidate how their interactions with tumor cells impact aggressiveness of metastatic tumors in bone in vitro methods are required. By employing a system in which BMMs and tumor cells are grown separately, yet share the media and exchange soluble factors, contribution of each cell type in the context of BMM-tumor cell relationship in the bone marrow can be investigated. Additional advantages of this system include the ability to study: 1) phenotypic changes in BMMs and tumor cells upon co-culture; 2) cell-specific modulation of protein and gene expression; and 3) effects on proliferation and cell survival. It is noteworthy, that this transwell co-culture system is not limited to BMMs and tumor cells and can be easily modified to include other components of bone marrow microenvironment (e.g., adipocytes, stromal cells, osteoblasts).
Pancreatic Acinar Cell 3-Dimensional Culture
Normal pancreatic acinar cells are difficult to maintain on traditional plastic culture surfaces due to their physical properties of housing large quantities of digestive enzymes and the formation of intercellular tight junctions and gap junctions (Apte and Wilson 2005; Rukstalis et al., 2003). However, placing primary acinar cells within a 3-dimensional matrix (3D-culture) maintains the cells for sufficient time so that they can be monitored for physiological changes to different stimuli. We have used a modified collagen 3D-culture system that has been adapted from Means et al. (2005) to model the very early events associated with pancreatic cancer development. In this model, KrasG12D-expressing pancreatic acinar cells, or wildtype acinar cells treated with EGFR-dependent growth factors (i.e., TGFα), convert to ductal cysts that mimic the acinar-to-ductal metaplasia (ADM) stage that precedes formation of Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Ductal Adenocarcinoma (PDAC) (Means et al., 2005; Shi et al., 2013).
Isolation of Mouse Embryo Fibroblasts
Preparation of primary cultures of embryo fibroblasts from genetically engineered mouse strains can provide a valuable resource for analyzing the consequences of genetic alterations at the cellular level. Mouse embryo fibroblasts (MEFs) have been particularly useful in cancer research, as they have facilitated the identification of the genetic changes that allow cells to overcome senescence and proliferate indefinitely in culture. The immortalized MEFs can then acquire additional mutations that lead to anchorage-independent growth and the ability to form tumors in mice. Recently we developed an MEF model system for analysis of the role of the tumor suppressor gene DLC1 in cellular transformation (Qian et al., 2012). In this communication we describe a protocol for the isolation of MEFs from day 13.5-day 14.5 mouse embryos. The MEFs obtained by this procedure are suitable for use in biochemical assays and for further genetic manipulations.
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