Cell Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 1003 Views Apr 5, 2022

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative, facultative intracellular bacterium, which causes gastrointestinal disorders in humans, and systemic, typhoid fever-like infections in mice. Our current knowledge regarding the involvement of cellular and humoral immunity in the defense from S. Typhimurium infections is largely based on animal models with attenuated strains. Cells of the innate immune system act as one of the first barriers in the defense from bacteria. We established a robust experimental model for the characterization of these cell types and their response during host-pathogen interactions. Therefore, this protocol focuses on the characterization of macrophages, monocytes, and neutrophils in the spleens of infected animals by employing multi-color flow cytometry.

0 Q&A 1216 Views Mar 20, 2022

Spontaneous DNA damage frequently occurs on the human genome, and it could alter gene expression by inducing mutagenesis or epigenetic changes. Therefore, it is highly desired to profile DNA damage distribution on the human genome and identify the genes that are prone to DNA damage. Here, we present a novel single-cell whole-genome amplification method which employs linear-copying followed by a split-amplification scheme, to efficiently remove amplification errors and achieve accurate detection of DNA damage in individual cells. In comparison to previous methods that measure DNA damage, our method uses a next-generation sequencing platform to detect misincorporated bases derived from spontaneous DNA damage with single-cell resolution.

0 Q&A 3118 Views Apr 20, 2021

Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here we present a method that allows for the analysis and selection of single fungal cells in high throughput by flow cytometry and fluorescence activated cell sorting (FACS), respectively. This protocol can be adapted for every fungal species that produces cells of up to 70 microns in diameter. After initial setting of the flow cytometry gates, which takes a single day, accurate single cell analysis and sorting can be performed. This method yields a throughput of thousands of cells per second. Selected cells can be subjected to downstream experiments to study single-cell behavior.

0 Q&A 8839 Views May 20, 2019
Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive and robust method that can optimize transfection efficiency based not only on expression of the target protein of interest but also on the uptake of the nucleic acids, can be an important tool in molecular biology. Herein, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency while overcoming limitations of prior established methods that quantify transfection efficiency.
0 Q&A 6854 Views May 20, 2018
The ability to conduct investigation of cellular transcription, signaling, and function at the single-cell level has opened opportunities to examine heterogeneous populations at unprecedented resolutions. Although methods have been developed to evaluate high-dimensional transcriptomic and proteomic data (relating to cellular mRNA and protein), there has not been a method to evaluate corresponding high-dimensional functionomic data (relating to cellular functions) from single cells. Here, we present a protocol to quantitatively measure the differentiation potentials of single human hematopoietic stem and progenitor cells, and then cluster the cells according to these measurements. High dimensional functionomic analysis of cell potential allows cell function to be linked to molecular mechanisms within the same progenitor population.
0 Q&A 8652 Views Jun 5, 2017
The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of pharmacologically inhibiting members of the bromodomain and extraterminal (BET) family of human bromodomain proteins (Filippakopoulos et al., 2010; Dawson et al., 2011; Delmore et al., 2011) that include BRD2, BRB3, BRD4 and BRDT. Small-molecule BET inhibitors, such as JQ1 (Filippakopoulos et al., 2010; Delmore et al., 2011), I-BET (Nicodeme et al., 2010), I-Bet151 (Dawson et al., 2011), and MS417 (Zhang et al., 2012) successfully activate HIV transcription and reverse viral latency in clonal cell lines and certain primary T-cell models of latency. To identify the mechanism by which BET proteins regulate HIV-1 latency, we utilized small hairpin RNAs (shRNAs) that target BRD2, BRD4 and Cyclin T1, which is a component of the critical HIV-1 cofactor positive transcription elongation factor b (P-TEFb) and interacts with BRD2, and tested them in the CD4+ J-Lat A2 and A72 cell lines. The following protocol describes a flow cytometry-based method to determine the amount of transcriptional activation of the HIV-1 LTR upon shRNA knockdown. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines.
0 Q&A 10209 Views May 20, 2017
The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral treatments with latency-purging strategies may accelerate the depletion of latent reservoirs and lead to a cure (Geeraert et al., 2008). Current strategies to reactivate HIV-1 from latency include use of prostratin, a non-tumor-promoting phorbol ester (Williams et al., 2004), BET inhibitors (Filippakopoulos et al., 2010; Delmore et al., 2011), and histone deacetylase (HDAC) inhibitors, such as suberoylanilidehydroxamic acid (i.e., SAHA or Vorinostat) (Kelly et al., 2003; Archin et al., 2009; Contreras et al., 2009; Edelstein et al., 2009). As the mechanisms of HIV-1 latency are diverse, effective reactivation may require combinatorial strategies (Quivy et al., 2002). The following protocol describes a flow cytometry-based method to quantify transcriptional activation of the HIV-1 long terminal repeat (LTR) upon drug treatment. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines that contain a GFP cassette. J-Lats that contain a different reporter, for example Luciferase, can be treated with drugs as described but have to be analyzed differently.



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