2014
DOI: 10.1371/journal.pone.0111437
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Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

Abstract: Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low … Show more

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Cited by 26 publications
(32 citation statements)
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“…We found SNAT1 protein to be poorly expressed in resting primary human CD4+ T cells but dramatically induced following mitogenic T cell stimulation ( Figures 3 F, lanes 2–5, and 3 G, panel 2). Furthermore, Vpu depletes SNAT1 from activated primary human CD4+ T cells both in the context of viral infection ( Figures S4 D and S4E) and as a single gene in transduced cells purified by Antibody-Free Magnetic Cell Sorting (AFMACS; Figures 3 G, panel 5, 3 H, lane 5, and S4 F) ( Matheson et al., 2014 ).…”
Section: Resultsmentioning
confidence: 99%
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“…We found SNAT1 protein to be poorly expressed in resting primary human CD4+ T cells but dramatically induced following mitogenic T cell stimulation ( Figures 3 F, lanes 2–5, and 3 G, panel 2). Furthermore, Vpu depletes SNAT1 from activated primary human CD4+ T cells both in the context of viral infection ( Figures S4 D and S4E) and as a single gene in transduced cells purified by Antibody-Free Magnetic Cell Sorting (AFMACS; Figures 3 G, panel 5, 3 H, lane 5, and S4 F) ( Matheson et al., 2014 ).…”
Section: Resultsmentioning
confidence: 99%
“…To identify endogenous SNAT1 substrates in primary human CD4+ T cells in an unbiased fashion, we combined an “activation-rest” strategy for shRNA knockdown ( Monroe et al., 2014 ) with Consumption and Release (CoRe) metabolomics ( Jain et al., 2012 ) ( Figure 5 A). Pure populations of transduced cells expressing control or SNAT1-specific shRNAs were generated by AFMACS ( Figures 5 B and S6 A) ( Matheson et al., 2014 ). After resting for 7–10 days, control and SNAT1-depleted cells were re-stimulated using CD3/CD28 Dynabeads.…”
Section: Resultsmentioning
confidence: 99%
“…For lentiviral shRNA-mediated knockdown of EloB ( TCEB2 ), EloC ( TCEB1) and CBFβ ( CBFB ) in 293T cells, hairpins were cloned into pHRSIREN-PGK-hygro (related to pHRSIREN-PGK-SBP-ΔLNGFR-W, but expressing hygromycin resistance (Matheson et al, 2014). The following oligonucleotides were inserted using BamHI-EcoRI (only top oligonucleotides are shown).…”
Section: Methodsmentioning
confidence: 99%
“…This construct was developed based on one initially designed for purification of transgenic T cells in suspension (Matheson, Peden, & Lehner, 2014), and it is included in Addgene cat. no.…”
Section: Selection and Enrichment Of Transgenic Cell Populationsmentioning
confidence: 99%