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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.

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Cell line controlled ratio sorts and subsequent WBs support validity of method to enrich for cell subpopulations.(a) Representative cell populations pre-FACS and histograms collected during FACS. Ab-labeled, TUBB3+ (green) population quantified. (b) Representative images of post-FACS, collected cell populations. (c) Representative WB of post-FACS, collected cell populations for TUBB3 expression. Densitometry-based quantification of TUBB3-positive expression from three, independent experiments (mean ± s.d.). One-way ANOVA was performed to determine significance among all control and enrichment groups (unlike letters are significantly different from each other, P < 0.04). Scale bars, 200 μm.
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f2: Cell line controlled ratio sorts and subsequent WBs support validity of method to enrich for cell subpopulations.(a) Representative cell populations pre-FACS and histograms collected during FACS. Ab-labeled, TUBB3+ (green) population quantified. (b) Representative images of post-FACS, collected cell populations. (c) Representative WB of post-FACS, collected cell populations for TUBB3 expression. Densitometry-based quantification of TUBB3-positive expression from three, independent experiments (mean ± s.d.). One-way ANOVA was performed to determine significance among all control and enrichment groups (unlike letters are significantly different from each other, P < 0.04). Scale bars, 200 μm.

Mentions: The ability to sort cell populations based on our intracellular target TUBB3 was evaluated by pre- and post-FACS fluorescence imaging and WB. In addition to TUBB3+ (SH-SH5Y cells only) and TUBB3− (SK-N-MC cells only) controls, cell lines were mixed together in a ratio of 25% SH-SY5Y cells: 75% SK-N-MC cells (25:75) to artificially establish a well-defined, heterogeneous population. As expected, imaging showed SH-SY5Y cells were primarily TUBB3+, SK-N-MC cells were primarily TUBB3−, and 25:75 cells were 27% TUBB3+ (Fig. 2a). All cell groups exhibited limited debris when assessing their forward and side scatter plots (Fig. 3a). Post-FACS, TUBB3 expression for cell groups remained consistent for all iterations of the experiment. SH-SY5Y cells had a single, primarily positive peak; SK-N-MC cells had a single, primarily negative peak; and 25:75 cells had two distinct peaks in which the positive peak (TUBB3+) had approximately one quarter of total events collected, indicating the cell separation worked as anticipated (Fig. 2a). Based on flow cytometric analysis, SH-SY5Y cells had a 20 ± 11% false negative rate, and SK-N-MC cells had a 7 ± 7% false positive rate. 25:75 samples had lower false positive and false negative rates (~3%) than those of single cell type samples. After sorting, fluorescence imaging of collected cells further corroborated these findings, showing that SH-SY5Y cells and TUBB3+ enrichment groups were primarily TUBB3+; both SK-N-MC cells and TUBB3− enrichment groups were primarily TUBB3−; and 25:75 unsorted group was 29% TUBB3+ (Fig. 2b).


Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations
Cell line controlled ratio sorts and subsequent WBs support validity of method to enrich for cell subpopulations.(a) Representative cell populations pre-FACS and histograms collected during FACS. Ab-labeled, TUBB3+ (green) population quantified. (b) Representative images of post-FACS, collected cell populations. (c) Representative WB of post-FACS, collected cell populations for TUBB3 expression. Densitometry-based quantification of TUBB3-positive expression from three, independent experiments (mean ± s.d.). One-way ANOVA was performed to determine significance among all control and enrichment groups (unlike letters are significantly different from each other, P < 0.04). Scale bars, 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036045&req=5

f2: Cell line controlled ratio sorts and subsequent WBs support validity of method to enrich for cell subpopulations.(a) Representative cell populations pre-FACS and histograms collected during FACS. Ab-labeled, TUBB3+ (green) population quantified. (b) Representative images of post-FACS, collected cell populations. (c) Representative WB of post-FACS, collected cell populations for TUBB3 expression. Densitometry-based quantification of TUBB3-positive expression from three, independent experiments (mean ± s.d.). One-way ANOVA was performed to determine significance among all control and enrichment groups (unlike letters are significantly different from each other, P < 0.04). Scale bars, 200 μm.
Mentions: The ability to sort cell populations based on our intracellular target TUBB3 was evaluated by pre- and post-FACS fluorescence imaging and WB. In addition to TUBB3+ (SH-SH5Y cells only) and TUBB3− (SK-N-MC cells only) controls, cell lines were mixed together in a ratio of 25% SH-SY5Y cells: 75% SK-N-MC cells (25:75) to artificially establish a well-defined, heterogeneous population. As expected, imaging showed SH-SY5Y cells were primarily TUBB3+, SK-N-MC cells were primarily TUBB3−, and 25:75 cells were 27% TUBB3+ (Fig. 2a). All cell groups exhibited limited debris when assessing their forward and side scatter plots (Fig. 3a). Post-FACS, TUBB3 expression for cell groups remained consistent for all iterations of the experiment. SH-SY5Y cells had a single, primarily positive peak; SK-N-MC cells had a single, primarily negative peak; and 25:75 cells had two distinct peaks in which the positive peak (TUBB3+) had approximately one quarter of total events collected, indicating the cell separation worked as anticipated (Fig. 2a). Based on flow cytometric analysis, SH-SY5Y cells had a 20 ± 11% false negative rate, and SK-N-MC cells had a 7 ± 7% false positive rate. 25:75 samples had lower false positive and false negative rates (~3%) than those of single cell type samples. After sorting, fluorescence imaging of collected cells further corroborated these findings, showing that SH-SY5Y cells and TUBB3+ enrichment groups were primarily TUBB3+; both SK-N-MC cells and TUBB3− enrichment groups were primarily TUBB3−; and 25:75 unsorted group was 29% TUBB3+ (Fig. 2b).

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 &beta;-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.


Related in: MedlinePlus