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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.


Related in: MedlinePlus

Methodology schematic.(1) Isolate tissue of interest and dissociate cells; (2) Fix samples in suspension with 10% formalin; (3) Label cells with a specific intracellular marker for the cell population of interest and select for cells using FACS; (4) Extract protein from collected samples, and run protein assays. Ab, antibody.
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f5: Methodology schematic.(1) Isolate tissue of interest and dissociate cells; (2) Fix samples in suspension with 10% formalin; (3) Label cells with a specific intracellular marker for the cell population of interest and select for cells using FACS; (4) Extract protein from collected samples, and run protein assays. Ab, antibody.

Mentions: Brown University’s Institutional Animal Care and Use Committee approved all protocols related to primary cell isolation (IACUC #1409000090A002), and the methods were carried out in accordance with the approved protocols. Whole brains were isolated from postnatal day 1–2 CD rats (Charles River). Cell isolation followed a modified BrainBits protocol49 (Fig. 5, Part 1). Briefly, meninges were removed from whole brains while submerged in Hibernate A buffer (BrainBits) supplemented with 1X B27 (Invitrogen) and 0.5 mM Gluta-Max. Each brain was minced into ~2 mm3 pieces and digested in 2 mg/mL papain (BrainBits) solution dissolved in Hibernate A without calcium buffer (BrainBits) at 30 °C for 30 minutes. After removing the papain solution, tissues were titurated 25 times with a fire polished Pasteur pipette in Hibernate A/B27 buffer. The cell suspension was centrifuged for 5 minutes at 150 × g and was resuspended in culture medium composed of Neurobasal A medium (Invitrogen) supplemented with 1X B27, 0.5 mM Gluta-Max, and 1X penicillin/streptomycin. To remove cellular debris, resuspended cells were passed through a 40 μm cell strainer, centrifuged for 5 minutes at 150 × g, and resuspended in culture medium. Cell straining, centrifugation, and resuspension were repeated once more. Cell viability and yields were calculated by using a trypan blue exclusion assay (HyClone, Thermo Scientific). On average, cell viability was 97% (±1%), and cell yields were 37 × 106 (±3 × 106) cells per pup.


Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations
Methodology schematic.(1) Isolate tissue of interest and dissociate cells; (2) Fix samples in suspension with 10% formalin; (3) Label cells with a specific intracellular marker for the cell population of interest and select for cells using FACS; (4) Extract protein from collected samples, and run protein assays. Ab, antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Methodology schematic.(1) Isolate tissue of interest and dissociate cells; (2) Fix samples in suspension with 10% formalin; (3) Label cells with a specific intracellular marker for the cell population of interest and select for cells using FACS; (4) Extract protein from collected samples, and run protein assays. Ab, antibody.
Mentions: Brown University’s Institutional Animal Care and Use Committee approved all protocols related to primary cell isolation (IACUC #1409000090A002), and the methods were carried out in accordance with the approved protocols. Whole brains were isolated from postnatal day 1–2 CD rats (Charles River). Cell isolation followed a modified BrainBits protocol49 (Fig. 5, Part 1). Briefly, meninges were removed from whole brains while submerged in Hibernate A buffer (BrainBits) supplemented with 1X B27 (Invitrogen) and 0.5 mM Gluta-Max. Each brain was minced into ~2 mm3 pieces and digested in 2 mg/mL papain (BrainBits) solution dissolved in Hibernate A without calcium buffer (BrainBits) at 30 °C for 30 minutes. After removing the papain solution, tissues were titurated 25 times with a fire polished Pasteur pipette in Hibernate A/B27 buffer. The cell suspension was centrifuged for 5 minutes at 150 × g and was resuspended in culture medium composed of Neurobasal A medium (Invitrogen) supplemented with 1X B27, 0.5 mM Gluta-Max, and 1X penicillin/streptomycin. To remove cellular debris, resuspended cells were passed through a 40 μm cell strainer, centrifuged for 5 minutes at 150 × g, and resuspended in culture medium. Cell straining, centrifugation, and resuspension were repeated once more. Cell viability and yields were calculated by using a trypan blue exclusion assay (HyClone, Thermo Scientific). On average, cell viability was 97% (±1%), and cell yields were 37 × 106 (±3 × 106) cells per pup.

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.


Related in: MedlinePlus