An auxiliary kit for detecting intracellular calcium ion content

Abstract

This invention provides an auxiliary kit for detecting intracellular calcium ion content, consisting of a concentrated culture medium additive and an auxiliary solvent. It improves the sensitivity and precision of the calcium ion fluorescent probe method for detecting intracellular calcium ion influx by maintaining the physiological function of the test cells, preventing premature hydrolysis of the fluorescent probe precursor, improving the solubility and stability of the fluorescent probe precursor, and inhibiting the leakage of activated fluorescent probes. Changes in intracellular calcium ions participate in various cellular physiological activities and are commonly detected in drug development, basic biological research, and other fields. The auxiliary kit provided by this invention can help researchers measure intracellular calcium ion influx more conveniently and accurately, obtaining more reliable results.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and relates to a method for detecting intracellular calcium ion influx, specifically an auxiliary method for detecting intracellular calcium ion influx using a calcium ion fluorescent probe. Background Technology

[0002] Various cellular physiological activities can cause changes in intracellular calcium ion concentration, including alterations in transmembrane potential, binding of various receptors (primarily membrane receptors) to ligands, and the opening or closing of ion channels. These cellular physiological activities are involved in various vital functions such as nerve signal transmission, muscle activity, and humoral regulation. Studying intracellular calcium ion influx, i.e., detecting intracellular calcium ion concentration, is an important method for understanding these physiological functions. The fluorescent probe method is the main method for detecting intracellular calcium ion concentration. Its principle is based on the fact that after a fluorescent probe chelates calcium ions, its fluorescence properties (quantum yield and / or fluorescence spectrum) change, which is then captured by a fluorescence detector. Because the fluorescent probe itself dissociates into anions at physiological pH, it is difficult to penetrate the cell membrane, and the extracellular calcium ion concentration is much higher than the intracellular calcium ion concentration. To increase the intracellular fluorescent probe concentration while avoiding interference from extracellular calcium ion binding, fluorescent probes are often made into inactive precursors that easily penetrate the cell membrane. Calcium ion fluorescent probe precursors are hydrolyzed intracellularly by esterases to form active fluorescent probes, and changes in their fluorescence properties reflect intracellular calcium ion intensity. However, cell culture media often require the addition of serum to maintain normal cell function, but serum contains a large number of persistently inactivated esterases that can prematurely hydrolyze calcium ion fluorescent probe precursors extracellularly, causing serious interference with detection. Current detection methods require changing the cell culture medium before detecting intracellular calcium ion concentration, which not only increases the number of steps and makes high-throughput difficult, but also has the potential to irritate cells and affect the accuracy of the detection results. To solve this problem, this invention provides an auxiliary kit for detecting intracellular calcium ion content. The concentrated culture medium additive can replace serum to maintain normal cell function while preventing premature extracellular hydrolysis of the calcium ion fluorescent probe precursor. The auxiliary solvent helps the highly lipid-soluble calcium ion fluorescent probe precursor to be better dispersed in the culture medium, while blocking the activity of cellular anion transporters to prevent the escape of the intracellularly hydrolyzed and activated calcium ion fluorescent probe, thus avoiding signal interference. Summary of the Invention

[0003] This invention provides an auxiliary kit consisting of two reagents for detecting intracellular calcium ion concentration using a fluorescent probe method. This kit can detect calcium ion influx induced by acetylcholine activation of the M3 receptor at the nanomolar level, while also improving the ease of operation of the method.

[0004] This invention is achieved through the following technical solution:

[0005] Concentrated culture medium additive, composed of the following components dispersed in water:

[0006]

[0007]

[0008] The concentrated culture medium additive as described in technical solution 1 is prepared by the following method:

[0009] The following water-soluble components or their soluble salts are dissolved in water sequentially, and then filtered through a water-based filter membrane with a pore size of 0.22 micrometers or 0.1 micrometers to remove bacteria, thus preparing a mother liquor of the water-soluble components.

[0010]

[0011]

[0012] The following fat-soluble components are mixed and added to water under high-speed stirring. Microemulsions are prepared by high-pressure homogenization or ultrasonic disruption, controlling the droplet size to below 200 nm. The microemulsion is then filtered and sterilized using a 0.22-micron pore size aqueous filter membrane to prepare the fat-soluble component mother liquor.

[0013]

[0014] The water-soluble component stock solution and the fat-soluble component stock solution are mixed, and then diluted with water to the predetermined concentration described in technical solution 1 to obtain a concentrated culture medium additive.

[0015] The auxiliary solvent is composed of the following components dissolved in an organic solvent:

[0016]

[0017] The organic solvent is one or a mixture of two or more of ethanol, isopropanol, dimethyl sulfoxide, dimethylacetamide, and N-methyl-2-pyrrolidone in any proportion.

[0018] The concentrated culture medium additive is used to maintain cell survival, and its addition amount in the cell culture medium is 0.1%-50% by volume.

[0019] The concentrated culture medium additive can protect the calcium ion fluorescent probe precursor from extracellular hydrolysis, and its addition amount in the cell culture medium is 0.1%-50% by volume.

[0020] The auxiliary solvent can dissolve the calcium ion fluorescent probe precursor and disperse it more evenly in the cell culture medium without producing visible turbidity or precipitation. Its addition amount in the cell culture medium is 0.01%-1% by volume.

[0021] The auxiliary solvent can prevent the intracellularly hydrolyzed and activated calcium ion fluorescent probe from escaping the cell through the cell's anion transporter, and its addition amount in the cell culture medium is 0.01%-1% by volume.

[0022] This solution provides an auxiliary kit for detecting intracellular calcium ion content, which can improve the detection effect of calcium ion fluorescent probes while simplifying the operation process, thus providing assurance and convenience for the detection of intracellular calcium ion concentration.

[0023] This invention improves the sensitivity and precision of the calcium ion fluorescent probe method for detecting cellular calcium ion influx by four aspects: maintaining the physiological function of the test cells, preventing premature hydrolysis of the fluorescent probe precursor, improving the solubility and stability of the fluorescent probe precursor, and inhibiting the leakage of activated fluorescent probes. Changes in intracellular calcium ions participate in various cellular physiological activities and are commonly used in drug development, basic biological research, and other fields. The auxiliary kit provided by this invention can help researchers measure cellular calcium ion influx more conveniently and accurately, obtaining more reliable results. Attached Figure Description

[0024] Figure 1 The calcium ion influx induced by acetylcholine was detected using a fluorescence microscope with the aid of the kit provided in this invention.

[0025] Figure 2 The calcium ion influx induced by carbacholine was detected using the FLIPR Penta high-throughput real-time fluorescence detection and analysis system with the aid of the kit provided in this invention.

[0026] Figure 3 The EC50 of carbacholine was detected using the FLIPR Penta high-throughput real-time fluorescence detection and analysis system with the aid of the kit provided in this invention.

[0027] Figure 4 The EC50 of carbacholine was detected using the FLIPR Penta high-throughput real-time fluorescence detection and analysis system with the aid of the kit provided in this invention. Detailed Implementation

[0028] Example 1: Preparation of concentrated culture medium additives 1

[0029] Disperse the following water-soluble components in 300 ml of water, and adjust the pH to 7.0 with 2 M NaOH.

[0030]

[0031]

[0032] Add the following ingredients to the above solution in sequence:

[0033]

[0034] Add 0.1M NaOH or HCl to pH 7.0, dilute with water to 500ml, and filter with a 0.22-micron pore size aqueous filter membrane to obtain a water-soluble mother liquor.

[0035] Mix the following fat-soluble ingredients and heat and stir in a 50°C water bath until completely dissolved.

[0036]

[0037]

[0038] Under high-speed stirring, the mixture of fat-soluble components was slowly added to 400ml of 50℃ warm water, ultrasonically broken down to a particle size of approximately 100nm, diluted with water to 500ml, and filtered through a 0.22-micron pore size aqueous filter membrane for sterilization to obtain the fat-soluble component mother liquor.

[0039] The water-soluble component mother liquor and the fat-soluble component mother liquor of the above are mixed in equal volumes to obtain a concentrated culture medium additive.

[0040] Example 2: Preparation of concentrated culture medium additives

[0041] Dissolve the following water-soluble components sequentially in 650 ml of water, then add sodium bicarbonate until the pH reaches 7.4:

[0042] Separately, disperse 500 mg of bovine insulin in 50 ml of water, add 1 M HCl until completely dissolved, and slowly add this solution to the above solution while stirring.

[0043] Add 0.1M NaOH or HCl to pH 7.0, dilute with water to 750ml, and filter with a 0.22-micron pore size aqueous filter membrane to obtain a water-soluble mother liquor.

[0044] Mix the following fat-soluble ingredients and heat and stir in a 50°C water bath until completely dissolved.

[0045]

[0046]

[0047] Under high-speed stirring, the mixture of fat-soluble components was slowly added to 180ml of 50℃ warm water, ultrasonically broken down to a particle size of approximately 100nm, diluted with water to 250ml, and filtered through a 0.22-micron pore size aqueous filter membrane for sterilization to obtain the fat-soluble component mother liquor.

[0048] The water-soluble component stock solution and the fat-soluble component stock solution were mixed at a volume ratio of 3:1 to obtain a concentrated culture medium additive.

[0049] Example 3: Preparation of auxiliary solvent 1

[0050] Dissolve 2.85g of probenecid and 20.0g of poloxamer 188 in 75ml of dimethylacetamide, and add dimethylacetamide to a total volume of 100ml to obtain an auxiliary solvent.

[0051] Example 4: Preparation of auxiliary solvent 2

[0052] Dissolve 2.85g of probenecid and 20.0g of poloxamer 188 in 75ml of N-methyl-2-pyrrolidone, and add N-methyl-2-pyrrolidone to a total volume of 100ml to obtain an auxiliary solvent.

[0053] Example 5: Imaging observation of calcium ion current fluorescence signal

[0054] Approximately 5 x 10 6 HEK293 cells (from WuXi AppTec) transfected with M3 acetylcholine receptors were washed twice with 10 ml of phenol red-free DMEM / F12 medium to thoroughly remove residual serum from the cell suspension. 9.9 ml of phenol red-free DMEM / F12 medium and 0.1 ml of the concentrated culture medium additive described in Example 1 were added, mixed thoroughly, and transferred to 80 μl per well of a 96-well plate. The plates were incubated overnight at 37°C in a carbon dioxide (air containing 5% carbon dioxide) incubator.

[0055] The calcium ion fluorescent probe Fluo4-AM (Aladdin, catalog number F140981) was dissolved in the auxiliary solvent described in Example 3 to obtain a Fluo4-AM solution with a final concentration of 1 mM. This Fluo4-AM solution was diluted 200 times its volume with a mixed medium consisting of 99% (v / v) phenol red-free DMEM / F12 medium and 1% (v / v) concentrated medium additive described in Example 1 to obtain the Fluo4-AM working solution. This Fluo4-AM working solution was added to the above-mentioned 96-well plate containing cells, 20 μl per well. The plate was incubated at 37°C for 0.5 h in a CO2 incubator.

[0056] Add acetylcholine to 100 nM in the above-mentioned 96-well plate containing cells, and immediately take a picture under a microscope. Figure 1 The results show that the auxiliary kit provided by this invention can detect the calcium ion influx induced by 100 nM acetylcholine.

[0057] Example 6: Determining the activity of acetylcholine receptor agonists by changes in intracellular calcium ion concentration

[0058] Approximately 5 x 10 6 HEK293 cells (from WuXi AppTec) transfected with M3 acetylcholine receptors were washed twice with 10 ml of phenol red-free DMEM / F12 medium to thoroughly remove residual serum from the cell suspension. 9.9 ml of phenol red-free DMEM / F12 medium and 0.1 ml of the concentrated culture medium additive described in Example 1 were added, mixed thoroughly, and transferred to 80 μl per well of a 96-well plate. The plates were incubated overnight at 37°C in a CO2 incubator.

[0059] Fluo4-AM (Aladdin, catalog number F140981) was dissolved in the auxiliary solvent described in Example 3 to obtain a Fluo4-AM solution with a final concentration of 2 mM. This Fluo4-AM solution was diluted 200-fold with a mixed medium consisting of 99% (v / v) phenol red-free DMEM / F12 medium and 1% (v / v) concentrated medium additive described in Example 1 to obtain the Fluo4-AM working solution. This Fluo4-AM working solution was added to the above-mentioned 96-well plate containing cells, 20 μl per well. The plate was incubated at 37°C for 2 h in a CO2 incubator.

[0060] Carbacholine was added to 0, 1, 10, and 100 nM in 96-well plates containing cells using the FLIPR Penta high-throughput real-time fluorescence detection and analysis system, and the fluorescence signal of Fluo4-calcium ion conjugate was read. Figure 2 The results show that the auxiliary kit provided by this invention can effectively detect carbacholine-induced calcium ion influx in the range of 1-100 nM.

[0061] Example 7: Determination of EC50 of carbacholine on M3 acetylcholine receptors by changes in intracellular calcium ion concentration

[0062] Approximately 3 x 10 6 HEK293 cells (from WuXi AppTec) transfected with M3 acetylcholine receptors were washed twice with 10 ml of phenol red-free DMEM / F12 medium to thoroughly remove residual serum from the cell suspension. 9.9 ml of phenol red-free DMEM / F12 medium and 0.1 ml of the concentrated culture medium additive described in Example 2 were added, mixed thoroughly, and transferred to 80 μl per well of a 96-well plate. The plates were incubated overnight at 37°C in a CO2 incubator.

[0063] Fluo4-AM (Aladdin, catalog number F140981) was dissolved in the auxiliary solvent described in Example 4 to obtain a Fluo4-AM solution with a final concentration of 2 mM. This Fluo4-AM solution was diluted 200-fold with a mixed medium consisting of 99% (v / v) phenol red-free DMEM / F12 medium and 1% (v / v) concentrated medium additive described in Example 2 to obtain the Fluo4-AM working solution. This Fluo4-AM working solution was added to the above-mentioned 96-well plate containing cells, 20 μl per well. The plate was incubated at 37°C for 2 h in a CO2 incubator.

[0064] Carbacholine was added to a predetermined concentration in a 96-well plate containing cells using the FLIPR Penta high-throughput real-time fluorescence detection and analysis system. The fluorescence signal of Fluo4-calcium ion conjugates was read, and the EC50 was calculated. Figure 3 ).

[0065] The commercially available FLIPR Calcium 6 Assay Kit (purchased from Molecular Devices) was used to measure and calculate the EC50 of the same batch of carbacholine using the same cell line (HEK293 cells transfected with the M3 acetylcholine receptor) as a model, following the standard operating procedure provided in the kit, through the FLIPR Penta high-throughput real-time fluorescence detection and analysis system. Figure 4 )

[0066] The EC50 data above demonstrates that the auxiliary reagent kit provided by this invention can achieve similar effects to imported commercial reagent kits while simplifying the operation steps.

Claims

1. An auxiliary kit for detecting intracellular calcium ion content, comprising concentrated culture medium additives and auxiliary solvents. The concentrated culture medium additive consists of the following components dispersed in water: L-alanyl-L-glutamine 0–400 mM (preferably 40–400 mM, more preferably 200–400 mM); Glutathione 3μM–300μM (preferably 30–300μM, more preferably 100–300μM); L-Ascorbic acid 2-phosphate 9 μM–0.9 mM (preferably 0.1–0.9 mM, more preferably 0.5–0.9 mM); Divalent manganese salts (e.g., manganese chloride) 0.25 nM–25 nM (preferably 2.5–25 nM, more preferably 10–25 nM); Metavanadate 2.5 nM–250 nM (25-250 nM, more preferably 100-250 nM); Selenite 29 nM–2.9 μM (0.29–2.9 μM, more preferably 10–29 μM); Ethanolamine 20 μM–2 mM (preferably 0.2–2 mM, more preferably 0.5–2 mM); Retinyl acetate 1 μM–100 μM (preferably 10–100 μM, more preferably 25–100 μM); α-Tocopherol 2μM-200μM (preferably 20–200μM, more preferably 50–200μM); Dexamethasone 0.2 μM–20 μM (preferably 2–20 μM, more preferably 5–20 μM); D-galactose 0.175–17.5 mM (preferably 1.75–17.5 mM, more preferably 5–17.5 mM); Medroxyprogesterone acetate 0.1 μM–10 μM (preferably 1–10 μM, more preferably 2.5–10 μM); Triiodothyronine 6nM–600nM (preferably 60–600nM, more preferably 150–600μM); L-carnitine 75 μM–7.5 mM (preferably 0.75–7.5 mM, more preferably 2–75 mM); Linolenic acid 0.25μM–25μM (preferably 2.5–25μM, more preferably 10–25μM); Taurine 0.1–10 mM (preferably 1–10 mM, more preferably 2.5–10 mM); Coenzyme Q10 0.25 μM–25 μM (preferably 2.5–25 μM, more preferably 10–25 μM); Cholesterol 25 μM–2.5 mM (preferably 0.25–2.5 mM, more preferably 1–2.5 mM); Oleic acid 20 μM–2 mM (preferably 0.2–2 mM, more preferably 0.5–2 mM); Arachidonic acid 4 μM–0.4 mM (preferably 0.04–0.4 mM, more preferably 1–4 mM); Bovine insulin 0–1000 mg / L (preferably 4–500 mg / L, more preferably 100–500 mg / L); Transferrin 7.5–750 mg / L (preferably 75–750 mg / L, more preferably 200–750 mg / L); Catalase 5–500 mg / L (preferably 50–500 mg / L, more preferably 100–500 mg / L); Superoxide dismutase 0.5–50 mg / L (preferably 5–50 mg / L, more preferably 15–50 mg / L); Hyaluronic acid 0–10 g / L (preferably 0–1 g / L, more preferably 0.2–1 g / L); Soy lecithin 10 mg / L–1 g / L (preferably 0.1–1 g / L, more preferably 0.25–1 g / L); Poloxamer (188) 100 mg / L–25 g / L (preferably 1–25 g / L, more preferably 5–25 g / L); Mouse sarcoma cell matrix (MatriGel) 0.1%-10% (v / v) (preferably 1%-10%, more preferably 2.5%-10%); HEPES 0–1M (preferably 0.1–1M, more preferably 0.25–1M); PIPES 0–1M (preferably 0.1–1M, more preferably 0.25–1M); Dimethylacetamide 0–100 g / L (preferably 10–75 g / L, more preferably 25–75 g / L); Ethanol 0–100 g / L (preferably 0–20 g / L, more preferably 0–5 g / L); The auxiliary solvent is composed of the following components dissolved in an organic solvent: Poloxamer (188) 10 g / L–250 g / L (preferably 50–250 g / L, more preferably 150–250 g / L); Probenecid 1mM–500mM (preferably 50–500mM, more preferably 200–500mM).

2. The auxiliary reagent kit according to claim 1, characterized in that: The organic solvent is one or a mixture of two or more of ethanol, isopropanol, dimethyl sulfoxide, dimethylacetamide, and N-methyl-2-pyrrolidone in any proportion.

3. The auxiliary reagent kit according to claim 1, characterized in that: The preparation method of the concentrated culture medium additive is as follows: 1) Dissolve one or more of the following water-soluble components or their soluble salts in water in the required proportion, filter through a water-based filter membrane with a pore size of 0.22-0.1 micrometers, collect the solution passing through the filter membrane, and prepare a mother liquor of water-soluble components; The water-soluble components are L-alanyl-L-glutamine, glutathione, L-ascorbic acid 2-phosphate, divalent manganese salt (e.g., manganese chloride), metavanadate, selenite, ethanolamine, D-galactose, triiodothyronine, L-carnitine, taurine, bovine insulin, transferrin, catalase, superoxide dismutase, hyaluronic acid, HEPES, PIPES, and mouse sarcoma cell matrix (MatriGel). 2) Add the following fat-soluble components to water in the required proportions under stirring, and prepare a microemulsion by high-pressure homogenization and / or ultrasonic disruption, controlling the droplet size to be below 200 nm. Filter the solution through a 0.22-micron pore size aqueous filter membrane, collect the solution passing through the filter membrane, and prepare the fat-soluble component mother liquor; the fat-soluble components are retinyl acetate, α-tocopherol, dexamethasone, medroxyprogesterone acetate, linolenic acid, coenzyme Q10, cholesterol, oleic acid, arachidonic acid, soybean lecithin, poloxamer 188, dimethylacetamide, and ethanol; 3) Mix the above-mentioned water-soluble component mother liquor with the fat-soluble component mother liquor, and dilute to the required concentration without adding water or by adding water again to obtain concentrated culture medium additive.

4. The application of the auxiliary kit according to any one of claims 1-3 in the process of detecting intracellular calcium ion concentration by the calcium ion fluorescent probe method.

5. The application according to claim 4, characterized in that: The concentrated culture medium additive can maintain cell survival, and its addition amount in the cell culture medium is 0.1%-50% by volume, preferably 0.5-5%, more preferably 0.5%-2%.

6. The application according to claim 4, characterized in that: The concentrated culture medium additive can protect the calcium ion fluorescent probe precursor from extracellular hydrolysis. Its addition amount in the cell culture medium is 0.1%-50% by volume, preferably 0.5-5%, more preferably 0.5%-2%.

7. The application according to claim 4, characterized in that: The auxiliary solvent can dissolve the calcium ion fluorescent probe precursor and disperse it more uniformly in the cell culture medium without producing visible turbidity or precipitation. The amount added to the cell culture medium is 0.01%-1% by volume, preferably 0.05%-1%, and more preferably 0.05%-0.5%.

8. The application according to claim 4, characterized in that: The auxiliary solvent can prevent the intracellularly hydrolyzed and activated calcium ion fluorescent probe from escaping from the cell through the cell's anion transporter. The amount of the auxiliary solvent added to the cell culture medium is 0.01%-1% by volume, preferably 0.05%-1%, and more preferably 0.05%-0.5%.

9. The application according to claim 4, characterized in that: The auxiliary kit is able to detect calcium ion influx induced by acetylcholine activation of M3 receptors at the nanomolar level.

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