RU2547594C2 - Method of detecting proteins in different cell types of mammals and human using fluorescein-5-isothiocyanate at microscopic level - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of detection of intracellular proteins using fluorescein-5-isothiocyanate (FITC) in various types of mammalian cells is provided, which are characterised by different levels of the synthetic process, using the method of confocal laser microscopy. The mammalian cells are fixed by paraformaldehyde (PFA) preventing the extraction of proteins in subsequent stages of staining, permeabilised with detergent, then stained for 2 h with FITC in a concentration of 1 mcg/ml. The cells are enclosed in Mowiol 4-88 adding DABCO (1,4-diazabicyclo[2.2.2] octane). The resulting preparations are used to analyse the localisation and the content of the protein component of the cells by confocal microscopy.

EFFECT: invention enables to obtain information and to investigate the location of proteins in the cells and to carry out a comparative analysis of protein content in cells with different levels of metabolism.

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Description

The invention relates to biotechnology and can be used to analyze the localization and content of the protein component in mammalian cells with various levels of synthetic processes.

It is known that fluorescein-5-isothiocyanate (FITC, chemical formula C ₂₁ H ₁₁ NO ₅ S; fluorescein-5-isothiocyanate) is a yellow-green fluorescent dye that is widely used to label proteins (primarily antibodies) and peptides in vitro (Harlow E., Lane D. Antibodies: A Laboratory Manual. // Cold Spring Harbor Laboratory Press. 1988. 726 p.). It has been established that in the composition of proteins and peptides FITC strongly binds to the α-amino groups of amino acids (Jullian M., Hernandez A., Maurras A., Puget K., Amblard M., Martinez J., Subra G. N-terminus FITC labeling of peptides on solid support: the truth behind the spacer. // Tetrahedron Letters. 2009. V. 50. P. 260-263).

It is also known that FITC is used as a dye for proteins for subsequent cell analysis by flow cytometry (Rodriguez-Sainz C., Valor L., Hernandez DC, Gil J., Carbone J., Pascual-Bernaldez M., Rodriguez-Alcantara F ., Martinez I., Vicario JL, Mallal S., Fernandez-Cruz E. Flow cytometry analysis with a new FITC-conjugated monoclonal antibody-3E12 for HLA-B * 57: 01 rapid screening in prevention of abacavir hypersensitivity in HIV-1 -infected patients. // HIV Clin. Trials. 2013. V. 14. N. 4. P. 160-164). However, this approach does not allow one to evaluate the location of the protein in individual cells, including oocytes and mammalian embryos.

It is also known that in vitro the protein content can be quantified by protein chemistry methods (for example, according to the Lowry method and the Bradford method) (Lowry O.N., Rosebrough NJ, Farr AL Protein measurement with the Folin phenol reagent. // J. Biol. Chem. 1951. V. 193. P. 265-275; Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. // Anal. Biochem. 1976. V. 72. P. 248-254). However, these approaches require the destruction (lysis) of cells and therefore do not provide information on the location of proteins inside the cells.

Thus, to date, there are no approaches to detect total intracellular proteins by microscopic analysis, despite their relevance in modern biology and medicine.

The objective of the invention is to develop a method for the qualitative and quantitative detection of proteins in cells of different types of mammals and humans, which are characterized by different levels of synthetic processes, microscopic analysis methods.

The problem is solved by staining cells with FITC dye (Biotium, USA) after their fixation with paraformaldehyde (PFA) and permeabilization with detergent Triton X-100. Fixation of PFA cells prevents the extraction of proteins at subsequent stages of staining, and permeabilization with a detergent facilitates dye access to intracellular proteins. PFA was diluted in PBS (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH ₂ PO ₄ and 8.1 mM Na ₂ HPO ₄ , pH 7.2-7.4). FITC is used at a working concentration of 1 μg / ml per FSB.

Staining conditions for somatic cells FITC describes Example 1.

Example 1

Normal mouse fibroblasts of the NIH / 3T3 line or human tumor cells of the HeLa and HEP-2 lines are cultured on glasses under standard conditions, i.e. in a culture medium containing 90% DMEM medium (modified Dulbecco medium, PanEco, Russia), 10% calf fetal serum (HyClone, USA), 2 mM L-glutamine and 250 IU of penicillin and streptomycin, at 37 ° C and 5% CO ₂ . Cells are washed in PBS and fixed in 3% PFA for 15 minutes. After washing in FSB (3 times for 10 min), the cells are treated with a 0.2% solution of Triton X-100 in FSB for 10 min at room temperature. Cells are washed from the detergent in PBS (3 times for 10 min), transferred to a solution of FITC at a concentration of 1 μg / ml for 60 minutes at room temperature. Cells were washed in three FSB shifts for 5 min and enclosed in Moviol 4-88 (Sigma-Aldrich, USA) with the addition of DABCO (1,4-diazobicyclo [2.2.2] octane, Sigma-Aldrich). Stained cells were examined using a LSM510 DuoScanMETA confocal microscope (Carl Zeiss, Germany) using a × 63 / 1.4 PlanNeofluar lens with the following parameters: excitation of FITC fluorescence by light with a wavelength of 488 nm, detection of FITC fluorescence - 520 nm. Typical cell staining options obtained under the conditions described above are shown in Fig. 1.

The method proposed in the application allows the detection of proteins also in oocytes and early mammalian embryos, as illustrated by Examples 2 and 3, respectively.

Example 2

Female inbred C57B1 / 6 mice 4-8 weeks old were injected with 7.5 IU (International Units) mare serum gonadotropin (HGFA, Sigma-Aldrich, USA) in 100 μl of sterile saline to stimulate oocyte maturation. After 46-48 hours, animals are killed by cervical dislocation. The ovaries are removed from the abdominal cavity and placed in M2 medium (Sigma-Aldrich, USA) containing 4 mg / ml bovine serum albumin. To prevent premature oocyte maturation, 10 μg / ml dbcAMP (dibutyril cAMP, sodium salt, Sigma-Aldrich, USA) is added to M2 medium. Oocytes were isolated under the control of a Stemi2000 stereomicroscope (Carl CarlZeiss ″, Germany), fixed with 3% PFA for 30 min at room temperature. Oocytes are washed with FSB (3 shifts of 15 min each) and incubated in 0.2% Triton X-100 for 20 min at room temperature. Oocytes are washed in PBS (3 times for 10 min) and placed in an FITC solution at a concentration of 1 μg / ml for 2 hours. After staining, oocytes are washed in PBS (3 times for 5 min) and transferred to plates for intravital observation. After washing in PBS, oocytes can be placed for 20 min in a DAPI stain solution (Sigma-Aldrich) diluted in PBS to a final concentration of 1 μg / ml to detect chromatin. Stained oocytes were examined using an LSM510 DuoScanMETA confocal microscope (CarlZeiss, Germany) using × 63 / 1.4 Plan Apochromat or × 40 / 0.75 Plan Neofiuar lenses.

Typical oocyte staining under the conditions described above is shown in Fig. 2.

Example 3

Embryos are prepared by introducing 7 ME HFA per mouse in 100 μl of sterile saline. 48 h after administration of HFFA, the female is given 7 ME of human chorionic gonadotropin (hCG), then the female is placed on the male. Mating is controlled by the presence of copulative plugs, and the age of the embryos is counted from the time of hCG injection. Unicellular embryos are removed from the oviducts after 19-26 hours, embryos at the stage of two blastomeres - 46-48 hours after the introduction of hCG. Fixation, staining of FITC and the study of embryos using a confocal laser microscope is performed as described in Example 2. A typical variant of staining of embryos is shown in Fig. 3.

To prove the specificity of the binding of FITC to proteins, enzymatic treatment of HeLa or NIH / 3T3 cells with proteinase K or RNase A is carried out, as described in Example 4.

Example 4

The cells of the first experimental group were placed in a proteinase K solution (Amresco, USA) at a concentration of 1 μg / ml on FSB and incubated for 15 minutes at room temperature. Control cells for the first experimental group are incubated at the same time in FSB. Cells of the second experimental group are placed in a solution of RNase A (Sigma-Aldrich) at a concentration of 2 mg / ml in PBS and incubated for 3 hours at 37 ° C. Controls are cells that incubate in the FSB at the same time at the same temperature. All control and all experimental cells stained with FITC as described in Example 1. The results confirming the sensitivity of FITC staining to proteinase K treatment and insensitivity to RNase A treatment are shown in Fig. 4.

Staining of intracellular proteins using FITC does not interfere with the immunocytochemical detection of specific (individual) proteins using specific antibodies to these proteins and staining of DAPI chromatin, as illustrated in Example 5.

Example 5

Normal mouse NIH / 3T3 mouse fibroblasts and human Hep-2 tumor cells were cultured as described in Example 1. Cells were washed in PBS and fixed in 3% PFA for 15 minutes. After washing in FSB (3 times for 10 min), the cells are treated with 0.2% Triton X-100 solution in FSB for 10 min at room temperature, washed from detergent in FSB (3 times for 10 min) and transferred to a solution of primary antibodies taken in optimal dilutions. Staining is carried out in a humid chamber for 60 minutes at room temperature. Primary antibodies are, for example, rabbit polyclonal antibodies to SC35 splicing factor (Abcam, USA), rabbit polyclonal antibodies to β-tubulin (Sigma-Aldrich) or murine monoclonal antibodies to nucleolar antigen A3 (Grigoriev A.A. ., Bulycheva T.I., Sheval E.V., Kalinina I.A., Zatsepina O.V. Cytological signs of suppression of total protein synthesis detected using a new monoclonal antibody. -346). Cells are washed in PBS (3 times for 10 min) and transferred to a solution of secondary antibodies taken in dilutions recommended by the manufacturer for 40 min at room temperature. Secondary antibodies are goat antibodies to rabbit or mouse immunoglobulins conjugated to Alexa Fluor 568 (Molecular Probes Inc., USA). Cells are washed in PBS (3 times for 10 min) and incubated with FITC for 60 min at room temperature. Cells are rapidly washed in PBS and placed in a 0.5 μg / ml DAPI solution for chromatin staining. Cells are washed in PBS (2 times for 5 min), enclosed in Moviol and examined under a microscope as described in Example 1. The results of staining of FITC cells and labeling with different antibodies are shown in Fig. 5.

The proposed method also allows a comparative analysis of the protein content in cells with different levels of metabolism, as illustrated by Examples 6 and 7.

Example 6

HeLa cells are incubated in the presence of cycloheximide - a universal inhibitor of protein synthesis (inhibitor concentration - 100 μg / ml) for 120 min. Inhibitor purchased from Sigma-Aldrich. Cells incubated with cycloheximide and control cells were stained with FITC and examined using an LSM510 DuoScanMETA confocal laser microscope as described in Example 1. Typical staining options are shown in Fig. 6, where (a) are cells of the control group and (b) are cells treated with cycloheximide. It is clearly seen that the incubation of cells with cycloheximide leads to a decrease in the intensity of their staining with FITC.

To quantify the change in fluorescence intensity in the control and experimental samples, use the software included with the confocal microscope or Image J image analysis software provided by the National Institute of Health (NIH, USA at http://rsbweb.nih.gov/ij /features.html). Both programs make it possible to construct profiles of the distribution of dye fluorescence intensities along a straight line drawn by the researcher through any part of the image, and allow obtaining similar results.

The results obtained using the Image J program are presented in Fig. 6 (c, d). Based on the values of the intensities of the total FITC fluorescence level in the control cells (Fig. 6, c) and cells incubated with cycloheximide (Fig. 6, d), it can be concluded that inhibition of protein synthesis during incubation of cells with cycloheximide leads to a decrease in the fluorescence intensity of FITC more than doubled.

Example 7

As the second model, NIH / 3T3 mouse fibroblasts were used, which were cultured for 2 days, as described in Example 1, and then transferred to DMEM containing 0.1% fetal calf serum for 72 hours. This led to the release of cells from proliferative cell cycle in the G0 period - a period characterized by a reduced metabolic rate compared to cells in the exponential growth phase (Geyer PK, Meyuhas O., Perry RP, Johnson LF Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts. // Mol Cell Biol. 1982. V. 2. No. 6. P.685-693). Cells of the control group (i.e., cells that are in the exponential growth phase) and cells that are in the G0 period are fixed, stained with FITC and examined using a LSM510 DuoScanMETA confocal laser microscope as described in Example 1. Images are processed as described in Example 6. Figure 7 (a, b) shows that the fluorescence brightness of cells in the G0 period is lower than in the control. Figures 7 (c, d) show that the fluorescence intensity of FITC in cells in the G0 period decreases almost twofold compared to control values.

The invention is illustrated by the following graphic materials:

Fig. 1. Staining of NIH / 3T3 mouse cells and human HeLa cells with FITC fluorescent dye. Left row - FITZ coloring; right row - corresponding phase-contrast images of cells. Scale line, 10 microns.

FITC stains nucleoli most clearly (indicated by arrowheads), which corresponds to published data on increased protein concentration in this particular cell compartment (Ahmad Y., Boisvert F.M., Gregor P., Cobley A., Lamond AI Nucleolar Proteome Database - 2008 update // Nucleic Acids Res. 2009. V. 37. P. 181-184). Significant amounts of protein are also detected in the nucleus (I) and cytoplasm (c), where the protein is uneven. Protein free zones probably correspond to intracytoplasmic vacuoles.

Fig. 2. Staining of preovulatory mouse oocytes FITC (a, d) and DAPI (b, c), (c, f) are phase-contrast images of oocytes. I am the core; c - cytoplasm; NPS - nucleolus-like body, or nucleolus. Scale line, 10 microns.

It is clearly seen that the oocyte nucleoli are stained with FITC. FITC nucleol staining does not prevent chromatin staining with DAPI dye (excitation of fluorescence with light with a wavelength of 359 nm, fluorescence detection of 461 nm) and detection of nucleoli in phase contrast mode.

Fig. 3. Staining of unicellular mouse embryos (a, b) and mouse embryos at the stage of two blastomeres (c, d) with FITC dye. Scale line, 10 microns.

It is clearly seen that the FITC allows the detection of intracellular localization of proteins in the cytoplasm (c) and nuclei (s) of early mammalian embryos. The highest concentration of proteins is characteristic of nucleolar precursors (PN).

Fig. 4. Staining of NIH / 3T3 FITC cells in the control (a, c) and after treatment with proteinase K (b) or RNase A (d), c - cytoplasm; I am the core; arrowheads - nucleoli. Scale line, 10 microns.

It can be seen that treatment with proteinase K weakens the intensity of staining of cells (especially nucleoli) of FITC (compare a and b), while incubation of cells with RNase A does not significantly reduce the intensity of their fluorescence (compare c and d).

Fig.5. Staining of NIH / 3T3 mouse cells (a-e) and human HEP-2 (g-i) with FITC dye and antibodies to the cytoplasmic microtubule protein β-tubulin (b), nuclear splicing factor SC-35 mRNA (e), nucleolar protein A3 (h); (c, e, and) - staining of cells with DAPI dye. c - cytoplasm; I am the core; arrowheads - nucleoli. Scale line, 10 microns.

It can be seen that FITC does not interfere with the immunocytochemical detection of proteins with localization inside the cytoplasm (b), nucleus (e) and nucleoli (h), as well as chromatin staining in mouse (c, f) and human (s) cells using DAPI. Numerous brightly colored heterochromatin blocks — chromocenters — are clearly visible in mouse cells.

Fig. 6. Staining of HeLa cells with FITC dye in control (a) and after treatment with cycloheximide at a concentration of 100 μg / ml for 120 minutes; (c, d) —FITC fluorescence intensity distribution profiles along an arbitrary straight line drawn through the cells in the control (c) and after treatment with cycloheximide (g). The horizontal axis is the distance (in pixels) from the beginning of the straight line (lower angle), the vertical axis is the FITC fluorescence intensity profiles (in arbitrary units, UE) along this straight line. White arrows in figures (a) and (b) indicate the position of sites with maximum fluorescence; corresponding indicators of fluorescence intensity are indicated in figures (c) and (d) by black arrows. Scale line, 10 microns.

It is seen that the incubation of cells with cycloheximide leads to a visual decrease in the fluorescence intensity (compare a and b). Figures (c) and (d) show that the maximum FITC fluorescence values in the control reach 80 UE, and after treatment with cycloheximide, they are about 25 UE, i.e. more than halved.

Fig. 7. FITC stain staining of proliferating NIH / 3T3 fibroblasts (a, control) and fibroblasts synchronized in the G0 period (b); (c, d) —FITC fluorescence intensity distribution profiles along an arbitrary line in the control and after synchronization in the G0 period, respectively. The horizontal axis is the distance (in pixels) from the beginning of the straight line (lower angle), the vertical axis is the FITC fluorescence intensity profiles (in arbitrary units, UE) along this straight line. The white arrows in figures (a) and (b) indicate the position of the areas with the maximum fluorescence intensity; corresponding indicators of fluorescence intensity are indicated in figures (c) and (d) by black arrows. Scale line, 10 microns.

It can be seen that cell synchronization in the G0 period leads to a visual decrease in the fluorescence intensity of FITC compared with asynchronously dividing cells (compare a and b). Figures (c) and (d) show that the maximum FITC fluorescence in asynchronous cells reaches 60 UE, and in the control cells 35 UE, i.e. decrease almost twice.

Claims (1)

A method for detecting proteins in different types of mammalian and human cells using a fluorescent dye fluorescein-5-isothiocyanate (FITC), comprising fixing the cells with 3% paraformaldehyde prepared in 0.1 M phosphate-buffered saline (PBS) for 15 minutes at room temperature, treating the cells with a 0.2% solution of Triton X-100 on FSB for 10 minutes at room temperature, staining the FITC cells for 2 hours at room temperature and imprisoning the cells in Moviol 4-88 (Sigma-Aldrich, USA) with the addition of DABCO (1,4-diazobicyclo [2.2.2] octane, Sigma-Aldrich) for subsequent analysis by confocal microscopy.

Images