Effective Flow Cytometric Phenotyping of Cells Using Minimal Amounts of Antibody

Sharma, Deepak; Eichelberg, Mark R.; Haag, Jill D.; Meilahn, Amanda L.; Muelbl, Matthew J.; Schell, Kathleen; Smits, Bart M. G.; Gould, Michael N.

doi:10.2144/0000113854

Cited by 11 publications

(8 citation statements)

References 14 publications

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“…The expression of nrf-2, p65, p21 and Manganese Super Oxide Dismutase (Mn-SOD) genes was normalized against that of a housekeeping gene, ȕ-actin, and was plotted as fold change with respect to control. Table 1 Gene analysed using FlowJo 7.6.5 (Treestar, Inc. Ashland) software [27]. For intracellular staining, the cells (3 x 10 6 ) were fixed with 2% paraformaldehyde, permeabilized with 0.05% Tween 20 (5 min) and 0.1% TritonX100 (3 min) and stained with specific antibodies (1µg/100µl, 30 min) and then stained with Hoechst 33342 (10µg/ml, 4 hr).…”

Section: Rna Isolation Cdna Synthesis and Quantitative Real Time Pcrmentioning

confidence: 99%

Amelioration of radiation-induced hematopoietic syndrome by an antioxidant chlorophyllin through increased stem cell activity and modulation of hematopoiesis

Suryavanshi

Sharma

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et al. 2015

Free Radical Biology and Medicine

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Section: Rna Isolation Cdna Synthesis and Quantitative Real Time Pcrmentioning

confidence: 99%

Amelioration of radiation-induced hematopoietic syndrome by an antioxidant chlorophyllin through increased stem cell activity and modulation of hematopoiesis

Suryavanshi

Sharma

Checker

et al. 2015

Free Radical Biology and Medicine

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“…The abundance of PNLhi or CD61hi subpopulations (Figure 5B) as well as the mean fluorescence intensities (not shown) of these stainings were not different between susceptible and resistant Mcs1a congenic animals. In addition, we looked at MMEC differentiation in WT and MD mice by FACS analysis, as described previously [42]. Like for the rat, we used antibodies against CD45 and CD31 to exclude hematopoietic and endothelial cells, respectively, and antibodies against CD24 and CD29 to quantify luminal and basal MMEC populations (Figure 5C).…”

Section: Resultsmentioning

confidence: 99%

“…Rat and mouse mammary epithelial cells (RMECs and MMECs) were prepared from LN-excised abdominal and adjacent inguinal mammary glands, as described previously [25], [42]. For phenotyping RMECs and MMECs, staining was done using the low-volume staining method to reduce antibody costs [42].…”

Section: Methodsmentioning

confidence: 99%

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The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

et al. 2013

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Genome-wide association studies have revealed that many low-penetrance breast cancer susceptibility loci are located in non-protein coding genomic regions; however, few have been characterized. In a comparative genetics approach to model such loci in a rat breast cancer model, we previously identified the mammary carcinoma susceptibility locus Mcs1a. We now localize Mcs1a to a critical interval (277 Kb) within a gene desert. Mcs1a reduces mammary carcinoma multiplicity by 50% and acts in a mammary cell-autonomous manner. We developed a megadeletion mouse model, which lacks 535 Kb of sequence containing the Mcs1a ortholog. Global gene expression analysis by RNA-seq revealed that in the mouse mammary gland, the orphan nuclear receptor gene Nr2f1/Coup-tf1 is regulated by Mcs1a. In resistant Mcs1a congenic rats, as compared with susceptible congenic control rats, we found Nr2f1 transcript levels to be elevated in mammary gland, epithelial cells, and carcinoma samples. Chromatin looping over ∼820 Kb of sequence from the Nr2f1 promoter to a strongly conserved element within the Mcs1a critical interval was identified. This element contains a 14 bp indel polymorphism that affects a human-rat-mouse conserved COUP-TF binding motif and is a functional Mcs1a candidate. In both the rat and mouse models, higher Nr2f1 transcript levels are associated with higher abundance of luminal mammary epithelial cells. In both the mouse mammary gland and a human breast cancer global gene expression data set, we found Nr2f1 transcript levels to be strongly anti-correlated to a gene cluster enriched in cell cycle-related genes. We queried 12 large publicly available human breast cancer gene expression studies and found that the median NR2F1 transcript level is consistently lower in ‘triple-negative’ (ER-PR-HER2-) breast cancers as compared with ‘receptor-positive’ breast cancers. Our data suggest that the non-protein coding locus Mcs1a regulates Nr2f1, which is a candidate modifier of differentiation, proliferation, and mammary cancer risk.

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“…For the 50 nm microbeads (Miltenyi) that we use, | m p | = 10 6 Bohr magnetons 33 . We assume the total number of particles per cell to be n = 10 4 particles, a conservative estimate, based on the 10 5 CD45 receptors per leukocyte 34 . The Stoke’s drag of a trapped cell is F d = 6πηrv, where η = 0.8 mPa*s is the viscosity of water and r is the radius of the cell.…”

Section: Methodsmentioning

confidence: 99%

A magnetic micropore chip for rapid (<1 hour) unbiased circulating tumor cell isolation and in situ RNA analysis

Bhagwat

Yee

et al. 2017

Lab Chip

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The use of microtechnology for the highly selective isolation and sensitive detection of circulating tumor cells has shown enormous promise. One challenge for this technology is that the small feature sizes – which are the key to this technology’s performance – can result in low sample throughput and susceptibility to clogging. Additionally, conventional molecular analysis of CTCs often requires cells to be taken off-chip for sample preparation and purification before analysis, leading to the loss of rare cells. To address these challenges, we have developed a microchip platform that combines fast, magnetic micropore based negative immunomagnetic selection (>10 mL/hr) with rapid on-chip in-situ RNA profiling (>100× faster than conventional RNA labeling). This integrated chip can isolate both rare circulating cells and cell clusters directly from whole blood and allow individual cells to be profiled for multiple RNA cancer biomarkers, achieving sample-to-answer in less than 1 hour for 10 mL of whole blood. To demonstrate the power of this approach, we applied our device to the circulating tumor cell based diagnosis of pancreatic cancer. We used a genetically engineered lineage-labeled mouse model of pancreatic cancer (KPCY) to validate the performance of our chip. We show that in a cohort of patient samples (N = 25) that this device can detect and perform in-situ RNA analysis on circulating tumor cells in patients with pancreatic cancer, even in those with extremely sparse CTCs (< 1 CTC / mL of whole blood).

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Effective Flow Cytometric Phenotyping of Cells Using Minimal Amounts of Antibody

Cited by 11 publications

References 14 publications

Amelioration of radiation-induced hematopoietic syndrome by an antioxidant chlorophyllin through increased stem cell activity and modulation of hematopoiesis

Amelioration of radiation-induced hematopoietic syndrome by an antioxidant chlorophyllin through increased stem cell activity and modulation of hematopoiesis

The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

A magnetic micropore chip for rapid (<1 hour) unbiased circulating tumor cell isolation and in situ RNA analysis

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