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Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations

View Article: PubMed Central - PubMed

ABSTRACT

Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.

No MeSH data available.


Heterogeneity found in neonatal rat brain cells underlines importance of subpopulation identification and enrichment for more useful/accurate protein characterization.(a) Representative images of primary, neonatal, rat brain cells cultured in 2D. Cell nuclei are labeled with DAPI (blue). Scale bar, 100 μm. (b) Representative image of TUBB3-immunostained, unsorted cells in suspension prior to enrichment and subsequent distribution of TUBB3-associated fluorescence levels (green). Scale bar, 200 μm. (c) Representative images of post-FACS, collected cells. TUBB3 (green) population was quantified. Scale bars, 200 μm. (d) IF images of primary neonatal rat brain cells cultured in 2D. P-tau (green), t-tau (red), and DAPI (blue) populations were quantified. Identification of cell types was based on tau expression and morphology. (1) Neuron; (2) Astrocyte; (3) Oligodendrocyte. Scale bar, 100 μm. (e) WB and quantification of post-FACS, collected cells.
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f4: Heterogeneity found in neonatal rat brain cells underlines importance of subpopulation identification and enrichment for more useful/accurate protein characterization.(a) Representative images of primary, neonatal, rat brain cells cultured in 2D. Cell nuclei are labeled with DAPI (blue). Scale bar, 100 μm. (b) Representative image of TUBB3-immunostained, unsorted cells in suspension prior to enrichment and subsequent distribution of TUBB3-associated fluorescence levels (green). Scale bar, 200 μm. (c) Representative images of post-FACS, collected cells. TUBB3 (green) population was quantified. Scale bars, 200 μm. (d) IF images of primary neonatal rat brain cells cultured in 2D. P-tau (green), t-tau (red), and DAPI (blue) populations were quantified. Identification of cell types was based on tau expression and morphology. (1) Neuron; (2) Astrocyte; (3) Oligodendrocyte. Scale bar, 100 μm. (e) WB and quantification of post-FACS, collected cells.

Mentions: Neonatal rat brain cells were used as a representative model of cellular heterogeneity found in primary tissues. To visualize the variety of cell types present in these primary samples, cells were cultured on poly-D-lysine (PDL)-coated plastic for 4 days, and immunostained for standard, brain cell type markers. Specifically, neurons (TUBB3+), astrocytes (glial fibrillary acidic protein; GFAP+), oligodendrocytes (oligodendrocyte marker 1; O1+), neural progenitor cells (NPC) (nestin+), and microglia (CD11b+) were identified (Fig. 4a). Fluorescence image analysis showed that approximately 44% of 2D cultured cells were TUBB3+. Quantification of the other brain markers showed 4% GFAP+, 12% Nestin+, 9% CD11b+, and 17% O1+ for a single, independent run. The extent of co-expression for specific markers in these cells was not evaluated.


Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations
Heterogeneity found in neonatal rat brain cells underlines importance of subpopulation identification and enrichment for more useful/accurate protein characterization.(a) Representative images of primary, neonatal, rat brain cells cultured in 2D. Cell nuclei are labeled with DAPI (blue). Scale bar, 100 μm. (b) Representative image of TUBB3-immunostained, unsorted cells in suspension prior to enrichment and subsequent distribution of TUBB3-associated fluorescence levels (green). Scale bar, 200 μm. (c) Representative images of post-FACS, collected cells. TUBB3 (green) population was quantified. Scale bars, 200 μm. (d) IF images of primary neonatal rat brain cells cultured in 2D. P-tau (green), t-tau (red), and DAPI (blue) populations were quantified. Identification of cell types was based on tau expression and morphology. (1) Neuron; (2) Astrocyte; (3) Oligodendrocyte. Scale bar, 100 μm. (e) WB and quantification of post-FACS, collected cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036045&req=5

f4: Heterogeneity found in neonatal rat brain cells underlines importance of subpopulation identification and enrichment for more useful/accurate protein characterization.(a) Representative images of primary, neonatal, rat brain cells cultured in 2D. Cell nuclei are labeled with DAPI (blue). Scale bar, 100 μm. (b) Representative image of TUBB3-immunostained, unsorted cells in suspension prior to enrichment and subsequent distribution of TUBB3-associated fluorescence levels (green). Scale bar, 200 μm. (c) Representative images of post-FACS, collected cells. TUBB3 (green) population was quantified. Scale bars, 200 μm. (d) IF images of primary neonatal rat brain cells cultured in 2D. P-tau (green), t-tau (red), and DAPI (blue) populations were quantified. Identification of cell types was based on tau expression and morphology. (1) Neuron; (2) Astrocyte; (3) Oligodendrocyte. Scale bar, 100 μm. (e) WB and quantification of post-FACS, collected cells.
Mentions: Neonatal rat brain cells were used as a representative model of cellular heterogeneity found in primary tissues. To visualize the variety of cell types present in these primary samples, cells were cultured on poly-D-lysine (PDL)-coated plastic for 4 days, and immunostained for standard, brain cell type markers. Specifically, neurons (TUBB3+), astrocytes (glial fibrillary acidic protein; GFAP+), oligodendrocytes (oligodendrocyte marker 1; O1+), neural progenitor cells (NPC) (nestin+), and microglia (CD11b+) were identified (Fig. 4a). Fluorescence image analysis showed that approximately 44% of 2D cultured cells were TUBB3+. Quantification of the other brain markers showed 4% GFAP+, 12% Nestin+, 9% CD11b+, and 17% O1+ for a single, independent run. The extent of co-expression for specific markers in these cells was not evaluated.

View Article: PubMed Central - PubMed

ABSTRACT

Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.

No MeSH data available.