Since we published our white paper on single cell sorting (available here) in Spring 2018 there have been several more articles that we believe merit attention..
1. Sorting for molecular analysis e.g. RNA-seq
2. Sorting for downstream cell culture and assay
WHERE TO BUY
1. Sorting for molecular analysis
Jackson, L, et al. "Incomplete inhibition of HIV infection results in more HIV infected lymph node cells by reducing cell death." eLife 7 (2018): e30134.
This work highlights a delicate balance that the infecting HIV virus aims to achieve by infecting as many new cells as possible while killing the orginating cell of de novo virions. It points to a potential risk of using therapeutics to treat partially resistant viral strains, that exacerbated infection and effects of HIV patients. To enumerate HIV DNA copies per cell, cells were single-cell sorted a BD Biosciences FACSAria III sorter. GFP-positive, DRAQ7-negative RevCEM clones were sorted 1 day post-infection into 96 well plates for downstream PCR analysis.
Govindan, S, Oberst, P., & Jabaudon, D. "In vivo pulse-labeling of isochronic cohorts of cells in the central nervous system using FlashTag." bioRxiv (2018): 286831.
A new protocol to label, track and isolate newborn cells in the CNS in vivo. CFSE labelling (termed FlashTag) of progenitors in the embryonic CNS persists post-natally such that cells can be analyzed ex vivo or in fixed tissue, for electrophysiology, or FACS-sorted for cell culture or (single-cell) RNA-sequencing. When cells were isolated by FACS sorting, DRAQ7 was used to exclude dead and damaged cells from collection, downstream use and analysis. Sorting was performed on the Beckman Coulter Moflo Astrios cell sorter.
Humbert, M, et al. "Intratumoral CpG-B Promotes Antitumoral Neutrophil, cDC, and T-cell Cooperation without Reprograming Tolerogenic pDC." Cancer research 78.12 (2018): 3280-3292.
In an effort to affect immune tolerance towards tumours i.t. CpG-B (a TLR-9 agonist) was found to aid the tumour recruitment of neutrophils as tumour-associated conventional dendritic cells (TA-cDC) but did not re-programme resident plasmacytoid dendritic cells (TA-pDC), which were refractory to the treatment. To understand the gene expression profiles of the TA-pDC and TA-cDC cells were sorted on the basis of phenotype and exclusion of dead cells (DRAQ7+). Gene expression confirmed that the TA-cDC have upregulated gene expression associated with antitumour T-cell immunity, in contrast to the down-modulation in TA-pDC. Sorting was performed on the BioRad S3 Sorter.
Klingler, E, et al. "A translaminar genetic logic for the circuit identity of intracortically-projecting neurons." bioRxiv (2018): 290395.
This work shows intracortically-projecting neurons (ICPN) have transcriptional identities primarily in accord with input-output relationships rather than time of origin or laminar position. It appears there are conserved circuit-related transcriptional programs across cortical layers and that these may be maintained circuit features through development and evolution. For single sorting for downstream RNA-sequencing, fresh tissue was micro-dissected from regions of interest, digested and sieved. Singlet cells, positive for Hoechst and negative for DRAQ7, were sorted on the Beckman Coulter Moflo Astrios cell sorter for cDNA synthesis.
2. Sorting for downstream cell culture and assay
Handgraaf, S, et al. "17-β Estradiol regulates proglucagon-derived peptide secretion in mouse and human α-and L cells." JCI insight 3.7 (2018).
This article investigates the potentially beneficial role of the estrogenic pathway and, specifically of ERβ agonists to prevent type 2 diabetes. Human islets were dissociated with accutase and α and β cells sorted according to staining or not with an antibody against α-cell surface marker HPa1, excluding doublets and dead cells (DRAQ7+). Cells were cultured independently, treated with test compounds and controls and then co-cultured and assayed for GLP-1 and Insulin release. Sorting was performed on the BioRad S3 Sorter.
DRAQ7's unique constellation of properties make it the choice when live-sorting cells for downstream analysis, to exclude dead and damaged cells without risk of contamination of the intact cells, of interference of polymerase function or of cell culture potential.
1. Sorting for molecular analysis
Jackson, L, et al. "Incomplete inhibition of HIV infection results in more HIV infected lymph node cells by reducing cell death." eLife 7 (2018): e30134.
This work highlights a delicate balance that the infecting HIV virus aims to achieve by infecting as many new cells as possible while killing the orginating cell of de novo virions. It points to a potential risk of using therapeutics to treat partially resistant viral strains, that exacerbated infection and effects of HIV patients. To enumerate HIV DNA copies per cell, cells were single-cell sorted a BD Biosciences FACSAria III sorter. GFP-positive, DRAQ7-negative RevCEM clones were sorted 1 day post-infection into 96 well plates for downstream PCR analysis.
Govindan, S, Oberst, P., & Jabaudon, D. "In vivo pulse-labeling of isochronic cohorts of cells in the central nervous system using FlashTag." bioRxiv (2018): 286831.
A new protocol to label, track and isolate newborn cells in the CNS in vivo. CFSE labelling (termed FlashTag) of progenitors in the embryonic CNS persists post-natally such that cells can be analyzed ex vivo or in fixed tissue, for electrophysiology, or FACS-sorted for cell culture or (single-cell) RNA-sequencing. When cells were isolated by FACS sorting, DRAQ7 was used to exclude dead and damaged cells from collection, downstream use and analysis. Sorting was performed on the Beckman Coulter Moflo Astrios cell sorter.
Humbert, M, et al. "Intratumoral CpG-B Promotes Antitumoral Neutrophil, cDC, and T-cell Cooperation without Reprograming Tolerogenic pDC." Cancer research 78.12 (2018): 3280-3292.
In an effort to affect immune tolerance towards tumours i.t. CpG-B (a TLR-9 agonist) was found to aid the tumour recruitment of neutrophils as tumour-associated conventional dendritic cells (TA-cDC) but did not re-programme resident plasmacytoid dendritic cells (TA-pDC), which were refractory to the treatment. To understand the gene expression profiles of the TA-pDC and TA-cDC cells were sorted on the basis of phenotype and exclusion of dead cells (DRAQ7+). Gene expression confirmed that the TA-cDC have upregulated gene expression associated with antitumour T-cell immunity, in contrast to the down-modulation in TA-pDC. Sorting was performed on the BioRad S3 Sorter.
Klingler, E, et al. "A translaminar genetic logic for the circuit identity of intracortically-projecting neurons." bioRxiv (2018): 290395.
This work shows intracortically-projecting neurons (ICPN) have transcriptional identities primarily in accord with input-output relationships rather than time of origin or laminar position. It appears there are conserved circuit-related transcriptional programs across cortical layers and that these may be maintained circuit features through development and evolution. For single sorting for downstream RNA-sequencing, fresh tissue was micro-dissected from regions of interest, digested and sieved. Singlet cells, positive for Hoechst and negative for DRAQ7, were sorted on the Beckman Coulter Moflo Astrios cell sorter for cDNA synthesis.
2. Sorting for downstream cell culture and assay
Handgraaf, S, et al. "17-β Estradiol regulates proglucagon-derived peptide secretion in mouse and human α-and L cells." JCI insight 3.7 (2018).
This article investigates the potentially beneficial role of the estrogenic pathway and, specifically of ERβ agonists to prevent type 2 diabetes. Human islets were dissociated with accutase and α and β cells sorted according to staining or not with an antibody against α-cell surface marker HPa1, excluding doublets and dead cells (DRAQ7+). Cells were cultured independently, treated with test compounds and controls and then co-cultured and assayed for GLP-1 and Insulin release. Sorting was performed on the BioRad S3 Sorter.
DRAQ7's unique constellation of properties make it the choice when live-sorting cells for downstream analysis, to exclude dead and damaged cells without risk of contamination of the intact cells, of interference of polymerase function or of cell culture potential.
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