A method for immunofluorescence staining of suspended MMQ cells based on PDL coating

Abstract

The application discloses a kind of based on PDL coating's suspended MMQ cell immunofluorescence staining method, it is related to cell biology experimental technical field, including preparation PDL coating cell crawl sheet, MMQ suspended cell is inoculated to the crawl sheet and is adherent culture and completes drug treatment, after cell fixation, blocking is carried out primary antibody and secondary antibody incubation, whole process uses shaking table oscillation washing to replace centrifugal washing, completes first round fluorescence signal amplification and is removed by antibody elution Antibody complex is combined, repeated incubation and color development process realizes multi-target cyclic staining, finally after cell nucleus re-staining, mounting is carried out fluorescence imaging detection;The application is realized by PDL coating and adherent fixation of suspended cell, and incubation and washing system are optimized, while supporting multiple antibody elution and cyclic staining, comprehensively improve the effect and economy of suspended cell immunofluorescence experiment.

Description

Technical Field

[0001] This invention relates to the field of cell biology experimental technology, specifically to an immunofluorescence staining method for PDL-coated suspension MMQ cells. Background Technology

[0002] Pituitary adenoma (often simply called pituitary tumor) is a common benign intracranial tumor originating from the anterior pituitary cells, accounting for approximately 10%–15% of intracranial tumors. Its pathological mechanism research and drug screening heavily rely on in vitro cell models. Among existing pituitary adenoma research systems, rat-derived MMQ cells and GH3 cells are the most widely used classic cell lines. MMQ cells are typical suspension-growing cells, while GH3 cells exhibit semi-suspension growth characteristics.

[0003] Immunofluorescence staining is a core experimental technique for the localization and semi-quantitative detection of cell proteins. Traditional immunofluorescence procedures for suspended cells require repeated centrifugation for cell collection and washing, which has several technical drawbacks: First, repeated centrifugation easily leads to a large loss of cells, resulting in low cell utilization, which is particularly limiting when used for precious cell samples or low-abundance target detection. Second, centrifugation easily causes cell aggregation and clumping, resulting in insufficient washing and unbound antibody residues that can lead to high non-specific background noise and poor fluorescence signal-to-noise ratio. Third, the incubation system for centrifugation is usually large in volume, consuming large amounts of expensive reagents such as primary antibodies and TSA signal amplification reagents, thus increasing experimental costs. Fourth, the cell spread uniformity is poor, and the slide preparation effect is unstable, affecting the reproducibility of experimental results and imaging quality.

[0004] Poly-D-Lysine (PDL) is a strong cationic linear polymer that can firmly adhere negatively charged cells to the surface of a carrier through electrostatic interactions, exhibiting excellent adhesion-promoting and anti-detachment properties. It is commonly used in adherent cell culture and immunohistochemical experiments. However, current technologies lack a complete PDL-coated immunofluorescence staining protocol for suspended pituitary adenoma cells such as MMQ, particularly lacking a standardized method that combines PDL coating for adhesion, small-volume incubation, shaker washing, and multicolor cyclic staining. This fails to effectively address the aforementioned challenges in suspended cell immunofluorescence experiments. Summary of the Invention

[0005] The purpose of this invention is to provide a PDL-coated suspension MMQ cell immunofluorescence staining method to solve the problems of serious cell loss, large consumption of expensive reagents, insufficient washing, high background noise, poor slide preparation effect, and difficulty in achieving multi-target cyclic staining in existing suspension cell immunofluorescence staining techniques.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] According to a first aspect of this disclosure, a method for immunofluorescence staining of PDL-coated suspension MMQ cells is proposed, comprising the following steps:

[0008] S1. After sterilization, the cell spreader is placed in the first culture plate, and poly-D-lysine working solution is added until the cell spreader is completely submerged. Coating incubation is then performed. After incubation, the cell spreader is washed, dried, and PDL-coated cell spreader is obtained for later use.

[0009] S2. Take MMQ cells in the logarithmic growth phase, seed them onto the PDL-coated cell slide, and culture them until the MMQ cells are completely attached to the surface of the PDL-coated cell slide. Then, treat them with drugs according to experimental requirements.

[0010] S3. Discard the culture medium, wash the PDL-coated cell slide, add fixation solution to fix the attached MMQ cells; wash the PDL-coated cell slide again, add blocking solution and block at room temperature.

[0011] S4. Remove the PDL-coated cell slide from the first culture plate and place it on a glass slide with the cell attachment side facing up. Add primary antibody working solution until it completely covers the cell surface and incubate with primary antibody. After incubation, transfer the PDL-coated cell slide to the second culture plate, add washing solution until the PDL-coated cell slide is submerged, and place the second culture plate on a shaker for shaking and washing. After washing, add secondary antibody working solution for secondary antibody incubation, and after incubation, shake and wash again.

[0012] S5. Add fluorescent working solution to the PDL-coated cell slide to cover the cell surface, incubate at room temperature to complete signal amplification, and then wash with shaking in a shaker.

[0013] S6. Add antibody elution buffer to the PDL-coated cell smear after the first round of staining and treat at room temperature to remove the bound antibody complex; after shaking and washing, repeat the blocking operation in step S3, the antibody incubation and shaking washing operation in step S4, and the fluorescence signal amplification and staining operation in step S5 in sequence to complete at least one round of multi-target immunofluorescence staining.

[0014] S7. The PDL-coated cell slides that have been fully stained are counterstained with cell nuclei, washed, mounted, and detected using a fluorescence imaging device.

[0015] Further, in step S1, the concentration of the poly-D-lysine working solution is 0.05–0.1 mg / mL; the coating incubation conditions are incubation at 37°C for 30 min.

[0016] Further, in step S1, the cell crawling sheet is a circular cell crawling sheet with a diameter of 14 mm; the first culture plate is a 24-well cell culture plate, with one cell crawling sheet placed in each well, and the volume of poly-D-lysine working solution added to each well is 200 μL.

[0017] Further, in step S1, after the coating incubation is completed, the cell crawling slide is washed three times with PBS buffer, each time for 5 minutes; after washing, the residual liquid in the well is discarded, the slide is air-dried overnight at room temperature, stored under sterile conditions, and sterilized by ultraviolet irradiation before use.

[0018] Further, in step S2, the seeding density of the MMQ cells is 1 × 10⁻⁶ per well. 5 After inoculation, the first culture plate is shaken horizontally to distribute the cells evenly, and then placed in an incubator for overnight culture until the MMQ cells are completely attached to the surface of the PDL-coated cell slide.

[0019] Furthermore, in step S3, the fixative is a 4% paraformaldehyde solution, and the fixation condition is room temperature fixation for 10 minutes; the blocking solution is incubated at room temperature for 15 minutes, and after blocking, there is no need to wash before proceeding directly to the antibody incubation step.

[0020] Further, in step S4, the volume of the primary antibody working solution added is 20 μL, enough to completely cover the cell surface of the 14 mm diameter PDL-coated cell smear; the primary antibody incubation conditions are room temperature incubation for 60 min.

[0021] Further, in step S4, the second culture plate is a 12-well cell culture plate; the shaking speed of the shaker is 60 rpm, the washing time is 5 min each time, and a total of 3 washes are performed; the washing solution is PBST buffer or PBS buffer.

[0022] Furthermore, in step S6, the room temperature treatment time of the antibody elution solution is 20 min; each round of cyclic staining corresponds to different target proteins and different emission wavelengths of fluorescent colorimetric reagents.

[0023] According to a second aspect of this disclosure, an immunofluorescence staining method based on PDL-coated suspended MMQ cells is also proposed for the immunofluorescence detection of suspended or semi-suspended pituitary tumor cells, wherein the pituitary tumor cells include GH3 cells.

[0024] Compared with existing technologies, this invention provides a PDL-coated immunofluorescence staining method for suspended MMQ cells. By using PDL-coated cell spreaders, suspended MMQ cells achieve stable adhesion, eliminating the need for centrifugation during cell collection. This significantly reduces cell mechanical damage and loss, improving cell utilization and spread uniformity. A small-volume incubation system is constructed using 14mm spreaders, requiring only approximately 20μL of working solution to completely cover the cell surface, greatly reducing the amount of expensive reagents such as primary antibodies and TSA signal amplification reagents, effectively lowering experimental costs. Simultaneously, low-speed shaking washing replaces traditional centrifugation, ensuring thorough and uniform contact between the washing solution and the cell surface, efficiently removing unbound antibodies and impurities, reducing non-specific background fluorescence, and significantly improving the fluorescence signal-to-noise ratio and imaging quality. This method is compatible with antibody elution and multi-round sequential incubation procedures, enabling cyclic staining of multiple target proteins on the same sample, increasing the amount of information acquired per sample. Furthermore, the operation is standardized, the conditions are mild, human error is minimal, and the experimental stability and reproducibility are excellent. It can be extended to the immunofluorescence detection of various suspended or semi-suspended pituitary tumor cells, such as GH3. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0026] Figure 1 This is a schematic diagram of the multicolor immunofluorescence staining process for suspended cells in this invention;

[0027] Figure 2 This image shows the results of multicolor immunofluorescence staining of MMQ cells obtained using the method of this invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] The main reagents and materials used in this embodiment are as follows:

[0030] PDL stock solution (5mg / mL), Beyotime, catalog number C0312;

[0031] 14mm round cell-crawling slides, adhesive glass slides, Shitai;

[0032] 24-well cell culture plates, 12-well cell culture plates, NEST;

[0033] F12 / DMEM basal medium, fetal bovine serum, penicillin-streptomycin antibiotics, cell culture grade;

[0034] Paraformaldehyde, Tween-20, and PBS buffer, biochemically analytical grade;

[0035] Primary antibodies: β-arrestin2 (immunoway), DRD2, MDM2;

[0036] TSA fluorescent colorimetric reagents: TSA520, TSA570, TSA650; DAPI nucleoside reagent, antibody elution buffer, blocking buffer, antibody dilution buffer, and immunofluorescence reagents.

[0037] Reagent preparation:

[0038] PDL working solution: Dilute 5 mg / mL PDL stock solution 100 times with PBS buffer to prepare a 0.05 mg / mL working solution. Prepare and use immediately. The applicable PDL working concentration range is 0.05–0.1 mg / mL. This example uses 0.05 mg / mL to minimize the impact on cell state.

[0039] PBST buffer: Add 1 mL Tween-20 to 10 L 1×PBS buffer, mix thoroughly, and store at room temperature.

[0040] Complete culture medium: Add 10% fetal bovine serum and 1% antibiotics to F12 / DMEM basal medium, mix well and store at 4°C in the dark.

[0041] Detailed operation steps:

[0042] Step 1: Place the 14mm cell slide into a culture dish containing 75% alcohol, ensuring the alcohol completely covers the slide for disinfection; remove the slide with sterile forceps and place it in a sterile culture dish to air dry naturally;

[0043] Use sterile forceps to transfer the dried cell smears into 24-well culture plates, placing one smear in each well; add PBS buffer to cover the smears and wash three times, 5 min each time; after washing, use a pipette to remove any remaining PBS from the wells, and add 200 μL of 0.05 mg / mL PDL working solution to each well, ensuring that the solution completely submerges the smears; transfer the 24-well plate to a 37°C, 5% CO2 cell culture incubator and incubate for 30 min;

[0044] Remove the 24-well plate, discard the PDL working solution in the wells, add PBS buffer to wash the cell crawling slides 3 times, 5 min each time; aspirate the residual liquid in the wells and air dry overnight at room temperature; store the prepared PDL-coated cell crawling slides in a sterile environment, and sterilize them with ultraviolet light for 30 min in a laminar flow hood before use.

[0045] Step 2: Take MMQ suspension cells in the logarithmic growth phase, gently pipette to prepare a homogeneous cell suspension, and after cell counting, dispense 1 × 10⁻⁶ cells per well. 5 Seed cells at a density of 1,000 cells onto PDL-coated cell slides in 24-well plates; after seeding, gently shake the plate back and forth and left and right in the horizontal direction to distribute the cells evenly on the surface of the slides; transfer the 24-well plate to a 37°C, 5% CO2 incubator for overnight culture, and proceed with subsequent processing after the MMQ cells have completely adhered to the surface of the slides.

[0046] Discard the old culture medium in the wells, add PBS buffer to cover the slides, and gently wash three times for 5 minutes each time. After aspirating the PBS, add serum-free culture medium to each well for starvation treatment for 6 hours. After starvation, administer the drug treatment: the control group was given serum-free culture medium containing an equal volume of DMSO solvent, and the drug treatment group was given serum-free culture medium containing 100 μM CAB. Incubate for another 30 minutes.

[0047] Step 3: After drug treatment, discard the drug solution in the wells and wash the slides three times with PBS buffer. Add 4% paraformaldehyde solution to each well and fix at room temperature for 10 min. After fixation, discard the paraformaldehyde, add PBST buffer to cover the slides, transfer the 24-well plate to a shaker, and wash three times with shaking at 60 rpm for 5 min each time.

[0048] After washing, discard the PBST, add blocking solution to completely cover the surface of the cells on the slide, and incubate at room temperature for 15 minutes. After blocking, discard the blocking solution, and proceed directly to the antibody incubation step without washing.

[0049] Step 4: Gently remove the PDL-coated cell slide from the 24-well plate using sterile forceps. If the slide is tightly adhered and difficult to remove, gently lift the edge of the slide with a sterile toothpick before using forceps. Place the slide with the cell-attached side facing up on an adhesive glass slide. Gently absorb excess liquid along the edge of the slide with filter paper, being careful not to let the filter paper touch the cell growth area. The filter paper can be replaced with absorbent, stiff paper; do not use soft, easily collapsing paper, as it will absorb water and adhere to the cell slide.

[0050] Dilute the β-arrestin2 primary antibody with antibody dilution buffer at a ratio of 1:100. Take 20 μL of the primary antibody working solution and drop it onto the surface of the cells on the climbing slide, ensuring that the liquid evenly covers all cells. Incubate at room temperature in the dark for 60 min.

[0051] After the primary antibody incubation is complete, use tweezers to transfer the smears into 12-well culture plates, placing one smear in each well; add PBS buffer to cover the smears, place the 12-well plate on a shaker, and wash three times at 60 rpm for 5 minutes each time.

[0052] Step 5: After washing, discard the PBS, add the corresponding species' fluorescent secondary antibody working solution to cover the slide, and incubate at room temperature in the dark for 20 minutes. After incubation, add PBS buffer, shake at 60 rpm for 3 times, 5 minutes each time.

[0053] Remove the slide with tweezers, place it on a glass slide with the cell side facing up, and blot away excess moisture from the edges with filter paper. Dilute the TSA520 fluorescent reagent with the matching reaction solution at a ratio of 1:200, add 20 μL of the working solution to the surface of the slide, and incubate at room temperature in the dark for 10 min to amplify the signal. The signal intensity can be observed under a fluorescence microscope. If the signal is weak, the reaction time can be extended appropriately, but not exceeding 20 min.

[0054] After the color development is complete, place the slide back into the 12-well plate, add PBS buffer, and wash three times with shaking at 60 rpm for 5 minutes each time.

[0055] Step 6: Remove the swab and place it on a glass slide. Use filter paper to absorb excess water. Add antibody elution buffer to completely cover the cell surface of the swab and incubate at room temperature for 20 minutes to thoroughly remove the bound antibody complexes.

[0056] After elution, the slides were placed back into the 12-well plate and washed three times with PBS for 5 minutes each time. Then, the blocking, primary antibody incubation, secondary antibody incubation, fluorescence development and washing steps were repeated for the second round of target staining: the primary antibody was DRD2, diluted 1:50, and incubated overnight at 4°C. The corresponding fluorescent reagent was TSA570, diluted 1:200.

[0057] After completing the second round of staining, antibody elution and washing were performed again, and the above operations were repeated for the third round of target staining: the primary antibody was MDM2, diluted 1:50, and incubated at room temperature for 60 min; the corresponding fluorescent reagent was TSA650, diluted 1:200.

[0058] Step 7: After all targets have been stained, discard the washing solution, add DAPI working solution (1:100 dilution) to cover the slide, and stain the nuclei at room temperature in the dark for 10 minutes. After observing the counterstaining effect under a microscope, wash the slide three times with PBST buffer, 5 minutes each time.

[0059] After washing, remove the slide with tweezers and blot dry the edges with filter paper. Add an appropriate amount of anti-fluorescence quenching mounting medium to a clean glass slide, place the slide cell side down on the mounting medium, gently press it flat to avoid air bubbles, and blot away any excess mounting medium with filter paper.

[0060] The prepared slides were placed under a laser scanning confocal microscope (LSM 800), and the corresponding fluorescence channels were selected for observation and photography to obtain multicolor immunofluorescence imaging results of MMQ cells.

[0061] This method has a standardized operating procedure and is applicable to immunofluorescence staining detection of other suspended or semi-suspended pituitary tumor cells, such as GH3, in addition to MMQ cells.

[0062] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for immunofluorescence staining of PDL-coated suspension MMQ cells, characterized in that, Includes the following steps: S1. After sterilization, the cell spreader is placed in the first culture plate, and poly-D-lysine working solution is added until the cell spreader is completely submerged. Coating incubation is then performed. After incubation, the cell spreader is washed, dried, and PDL-coated cell spreader is obtained for later use. S2. Take MMQ cells in the logarithmic growth phase, seed them onto the PDL-coated cell slide, and culture them until the MMQ cells are completely attached to the surface of the PDL-coated cell slide. Then, treat them with drugs according to experimental requirements. S3. Discard the culture medium, wash the PDL-coated cell slide, add fixation solution to fix the attached MMQ cells; wash the PDL-coated cell slide again, add blocking solution and block at room temperature. S4. Remove the PDL-coated cell slide from the first culture plate and place it on a glass slide with the cell attachment side facing up. Add primary antibody working solution until it completely covers the cell surface and incubate with primary antibody. After incubation, transfer the PDL-coated cell slide to the second culture plate, add washing solution until the PDL-coated cell slide is submerged, and place the second culture plate on a shaker for shaking and washing. After washing, add the secondary antibody working solution for incubation. After incubation, wash again by shaking on a shaker. S5. Add fluorescent working solution to the PDL-coated cell slide to cover the cell surface, incubate at room temperature to complete signal amplification, and then wash with shaking in a shaker. S6. Add antibody elution buffer to the PDL-coated cell smear after the first round of color development, and treat at room temperature to remove the bound antibody complex; After washing by shaking on a shaker, the blocking operation in step S3, the antibody incubation and shaking washing operation in step S4, and the fluorescence signal amplification and color development operation in step S5 are repeated in sequence to complete at least one round of multi-target immunofluorescence staining. S7. Counterstain the cell nuclei of the PDL-coated cell slides after all staining has been completed, wash and mount the slides, and then detect them using a fluorescence imaging device.

2. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S1, the concentration of the poly-D-lysine working solution is 0.05–0.1 mg / mL; the coating incubation conditions are incubation at 37°C for 30 min.

3. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S1, the cell crawling sheet is a circular cell crawling sheet with a diameter of 14 mm; the first culture plate is a 24-well cell culture plate, with one cell crawling sheet placed in each well, and the volume of poly-D-lysine working solution added to each well is 200 μL.

4. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S1, after the coating incubation is completed, the cell crawling slide is washed three times with PBS buffer for 5 minutes each time; after washing, the residual liquid in the well is discarded, the slide is air-dried overnight at room temperature, stored under sterile conditions, and sterilized by ultraviolet irradiation before use.

5. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S2, the seeding density of MMQ cells is 1 × 10⁻⁶ per well. 5 After inoculation, the first culture plate is shaken horizontally to distribute the cells evenly, and then placed in an incubator for overnight culture until the MMQ cells are completely attached to the surface of the PDL-coated cell slide.

6. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S3, the fixative is a 4% paraformaldehyde solution, and the fixation conditions are room temperature fixation for 10 minutes; the blocking solution is incubated at room temperature for 15 minutes, and after blocking, there is no need to wash before proceeding directly to the antibody incubation step.

7. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S4, the volume of the primary antibody working solution added is 20 μL, enough to completely cover the cell surface of the 14 mm diameter PDL-coated cell smear; the primary antibody incubation conditions are room temperature incubation for 60 min.

8. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S4, the second culture plate is a 12-well cell culture plate; the shaking speed of the shaker is 60 rpm, the washing time is 5 min each time, and a total of 3 washes are performed; the washing solution is PBST buffer or PBS buffer.

9. The method for immunofluorescence staining of PDL-coated suspension MMQ cells according to claim 1, characterized in that, In step S6, the room temperature treatment time of the antibody elution solution is 20 min; each round of cyclic staining corresponds to different target proteins and different emission wavelengths of fluorescent colorimetric reagents.

10. The application of the PDL-coated suspended MMQ cell immunofluorescence staining method according to claim 1 in the immunofluorescence detection of suspended or semi-suspended pituitary tumor cells, wherein the pituitary tumor cells include GH3 cells.

Images