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Identifying Breast Cancer Treatment Biomarkers Using Transcriptomics
Published in Shazia Rashid, Ankur Saxena, Sabia Rashid, Latest Advances in Diagnosis and Treatment of Women-Associated Cancers, 2022
Multiple computational methods (Scott et al., 2020) have been developed to identify cell-type-specific methylation signals using DNA methylation for whole-genome bisulfite sequencing both from reference-based and non-reference-based. This field is known as immunomethylomics and is part of the collective effort of epigenome-wide association studies (EWAS). Methods that don’t need cell sorting have also been developed (Rahmani et al., 2019).
Genetics at the Cell Level
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Valentina Lorenzi, Roser Vento-Tormo
The first protocols for the unbiased quantification of the transcriptome of single cells were developed in a single tube containing lysis buffer (Tang et al., 2009). Shortly after, multiplexing and robotics enabled the processing of hundreds of cells, which meant that cells could be processed in 96-well or 384-well plates (Baugh et al., 2001; Islam et al., 2011; Ramsköld et al., 2012; Jaitin et al., 2014; Zeisel et al., 2015). Whereas the first protocols isolated cells via micro-pipetting and placed them into a plate containing lysis buffer, it is now common to isolate them by fluorescent activated cell sorting (FACS). This method allows the user to isolate cells by protein surface markers selected on the basis of prior information about the cell type. Following lysis of the cells, conversion of RNA into cDNA and subsequent cDNA amplification are performed separately on each cell and the PCR products are sequenced via next-generation sequencing (NGS) (Figure 2.4, top panel).
The Immunomagnetic Manipulation of Bone Marrow
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
With the identification of the CD34 antigen cluster as a marker for a population of early hemopoietic progenitors, positive selection has become of particular interest. Civin et al.4 have described a procedure, involving chymopapain treatment, for the removal of beads from CD34-positive cells. Cells treated in such a way have been shown to be highly viable and can be recovered in sufficient quantities from marrow to allow reconstitution after ablative doses of cytotoxic agents. Removal of cells from microspheres prior to reinfusion into the patient is critical, otherwise they will be rapidly removed from the circulation by the reticuloendothelial (RE) system. While this approach is interesting, it is not universally applicable. Poor recoveries of neuroblasts from microspheres have been achieved by enzymatically attempting to dissociate bead/tumor cell complexes. Considerable work therefore remains to obtain a true magnetic cell sorter that allows the separation and recovery of essentially all of the targeted population in a highly viable state. Once such a system has been devised, it will prove both more effective and cheaper than the conventional approach to cell sorting, namely, the fluorescence-activated cell sorter.
Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs
Published in mAbs, 2022
Andre A. R. Teixeira, Sara D’Angelo, M. Frank Erasmus, Camila Leal-Lopes, Fortunato Ferrara, Laura P. Spector, Leslie Naranjo, Esteban Molina, Tamara Max, Ashley DeAguero, Katherine Perea, Shaun Stewart, Rebecca A. Buonpane, Horacio G. Nastri, Andrew R. M. Bradbury
Given the size of each of these libraries, we performed first and second rounds of selection using magnetic-assisted cell sorting (MACS) at antigen concentrations of 10 nM and 1 nM, respectively. This allowed us to label and sort a larger number of cells than would have been practical using a flow cytometer. For subsequent rounds, we used fluorescence-activated cell sorting (FACS) to enable more precise sorting of the cells of interest. After the first three rounds of equilibrium sorting, we performed two rounds of kinetic sorting with 4 hours of competition for the L3 and H1H2 libraries. Only one 4 h kinetic sort was performed for the L1L2 library, followed by a negative sort, where the population was only incubated with secondary reagents and negative cells were sorted, which was done out of concern that enrichment of polyreactive antibodies may occur in light of the weak positivity observed in the absence of antigen
Immunologic evaluation and genetic defects of apoptosis in patients with autoimmune lymphoproliferative syndrome (ALPS)
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Laura Casamayor-Polo, Marta López-Nevado, Estela Paz-Artal, Alberto Anel, Frederic Rieux-Laucat, Luis M. Allende
The molecular study of somatic mutations in the FAS gene is mandatory in patients with elevated DNTs and biomarkers and in whom a germline mutation has not been identified. The workflow for genetic testing of somatic mutations (ALPS-sFAS) differs from that for germline mutations (Figure 6). Instead of working with DNA extracted from a whole blood sample, it is necessary to extract DNA from DNTs. For this application, the amount of whole blood required is higher (approximately 15 mL) because of the need to isolate DNTs using magnetic-activated cell sorting separation (Double-negative T Cell Isolation Kit (Miltenyi Biotec GmbH, Germany). Reference: 130-092-614) or fluorescence-activated cell sorting (FACS). For magnetic-activated cell sorting, the isolation of DNTs is performed in a two-step procedure: firstly, the CD4 + CD8+ and CD56+ cells are depleted from peripheral blood mononuclear cells (PBMC) and secondly, a positive selection of TCRαβ + CD4-CD8- cells are retained. For FACS sorting, PBMCs are stained with the same monoclonal antibody cocktail as for DNT immunophenotyping. In both cases, cell purity analysis is essential to ensure that there are more than 90% of DNTs. Finally, it is important to mention that somatic mutations in the FAS gene are restricted mainly to exons 7, 8, and 9 (Figure 6(A)).
Single-cell RNA sequencing: An overview for the ophthalmologist
Published in Seminars in Ophthalmology, 2021
Elizabeth J. Rossin, Lucia Sobrin, Leo A. Kim
One of the most common approaches for classifying cell type is fluorescence-activated cell sorting (FACS), which can be used to identify and sort sub-populations of cells based on size, morphology and surface proteins with the use of fluorescently conjugated antibodies. These sub-populations can be further characterized by pooled RNA sequencing or functional assays. However, marker-based approaches are inherently constrained by the availability and choice of markers and by our knowledge of how markers define cell types. Even within a seemingly narrowly defined group of cells based on cell surface markers, there is likely heterogeneity in gene expression signature.3 More recently, mass cytometry has been employed, which involves cell characterization with antibodies labelled by heavy metal ions, and this has dramatically increased the number of proteins that can be assessed at one time by five to 10-fold.4 Still, it is challenging to assess the entire proteome all at once with flow cytometry.