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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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Protein extraction from formalin-fixed cells is comparable to fresh cells when lysing protocol includes high concentrations of Tris-HCl, anionic detergent SDS, and high heat.(a) Expression of TUBB3 (green) in cell lines SH-SY5Y and SK-N-MC. Scale bar, 200 μm. DAPI, 4′,6-diamino-2-phenylindole (blue). (b) Total protein isolated from formalin-fixed cells was compared to fresh cells under two different lysing conditions (4 °C, ice; 100 °C, boil). Data represented as mean ± standard deviation (s.d.) from four, independent experiments. Within cell lines, one-way ANOVAs were performed to determine significance (unlike letters are significantly different from each other, P < 0.02). (c) Protein quality assessed by Coomassie Blue stained PAGE gel for general banding and WB for POI.
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f1: Protein extraction from formalin-fixed cells is comparable to fresh cells when lysing protocol includes high concentrations of Tris-HCl, anionic detergent SDS, and high heat.(a) Expression of TUBB3 (green) in cell lines SH-SY5Y and SK-N-MC. Scale bar, 200 μm. DAPI, 4′,6-diamino-2-phenylindole (blue). (b) Total protein isolated from formalin-fixed cells was compared to fresh cells under two different lysing conditions (4 °C, ice; 100 °C, boil). Data represented as mean ± standard deviation (s.d.) from four, independent experiments. Within cell lines, one-way ANOVAs were performed to determine significance (unlike letters are significantly different from each other, P < 0.02). (c) Protein quality assessed by Coomassie Blue stained PAGE gel for general banding and WB for POI.

Mentions: A consistently reliable protocol for processing fresh and fixed samples was selected based on total protein isolation. To maintain a controlled biological system while validating protein extraction protocols, two cell lines were chosen based on their expression of the neuronal marker, TUBB3: SH-SY5Y (TUBB3+, “neuronal”) and SK-N-MC (TUBB3−, “non-neuronal”). As verified by immunofluorescence (IF), SH-SY5Y cells highly expressed TUBB3, while SK-N-MC cells had very low TUBB3 expression (Fig. 1a). Three different, commonly used protein extraction protocols, as inspired from FFPE literature, were evaluated based on Coomassie Blue stained general protein banding and WB242627. Results showed that a lysis buffer containing 300 mM Tris-HCl, 2% sodium dodecyl sulfate (SDS), and 2X protease and phosphatase inhibitor cocktail26 assured maximal protein extraction from fixed samples without significant protein aggregation (Supplementary Fig. S1).


Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations
Protein extraction from formalin-fixed cells is comparable to fresh cells when lysing protocol includes high concentrations of Tris-HCl, anionic detergent SDS, and high heat.(a) Expression of TUBB3 (green) in cell lines SH-SY5Y and SK-N-MC. Scale bar, 200 μm. DAPI, 4′,6-diamino-2-phenylindole (blue). (b) Total protein isolated from formalin-fixed cells was compared to fresh cells under two different lysing conditions (4 °C, ice; 100 °C, boil). Data represented as mean ± standard deviation (s.d.) from four, independent experiments. Within cell lines, one-way ANOVAs were performed to determine significance (unlike letters are significantly different from each other, P < 0.02). (c) Protein quality assessed by Coomassie Blue stained PAGE gel for general banding and WB for POI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036045&req=5

f1: Protein extraction from formalin-fixed cells is comparable to fresh cells when lysing protocol includes high concentrations of Tris-HCl, anionic detergent SDS, and high heat.(a) Expression of TUBB3 (green) in cell lines SH-SY5Y and SK-N-MC. Scale bar, 200 μm. DAPI, 4′,6-diamino-2-phenylindole (blue). (b) Total protein isolated from formalin-fixed cells was compared to fresh cells under two different lysing conditions (4 °C, ice; 100 °C, boil). Data represented as mean ± standard deviation (s.d.) from four, independent experiments. Within cell lines, one-way ANOVAs were performed to determine significance (unlike letters are significantly different from each other, P < 0.02). (c) Protein quality assessed by Coomassie Blue stained PAGE gel for general banding and WB for POI.
Mentions: A consistently reliable protocol for processing fresh and fixed samples was selected based on total protein isolation. To maintain a controlled biological system while validating protein extraction protocols, two cell lines were chosen based on their expression of the neuronal marker, TUBB3: SH-SY5Y (TUBB3+, “neuronal”) and SK-N-MC (TUBB3−, “non-neuronal”). As verified by immunofluorescence (IF), SH-SY5Y cells highly expressed TUBB3, while SK-N-MC cells had very low TUBB3 expression (Fig. 1a). Three different, commonly used protein extraction protocols, as inspired from FFPE literature, were evaluated based on Coomassie Blue stained general protein banding and WB242627. Results showed that a lysis buffer containing 300 mM Tris-HCl, 2% sodium dodecyl sulfate (SDS), and 2X protease and phosphatase inhibitor cocktail26 assured maximal protein extraction from fixed samples without significant protein aggregation (Supplementary Fig. S1).

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