CN103645332B - Multifunctional workstation for automatically detecting outflow of cholesterol and other analyte cells with high throughput - Google Patents

Multifunctional workstation for automatically detecting outflow of cholesterol and other analyte cells with high throughput Download PDF

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CN103645332B
CN103645332B CN201310702939.8A CN201310702939A CN103645332B CN 103645332 B CN103645332 B CN 103645332B CN 201310702939 A CN201310702939 A CN 201310702939A CN 103645332 B CN103645332 B CN 103645332B
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cell
platform
described
sample
workstation
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CN201310702939.8A
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CN103645332A (en
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杭渤
约翰别利茨基
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南京卡迪奥密生物技术有限公司
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Abstract

The invention discloses a multifunctional workstation for automatically detecting the outflow of cholesterol and other analyte cells with high throughput. The multifunctional workstation comprises a constant-temperature short-period incubator (1), a cell washing and sample loading platform (2), a sample filtering platform (3), a sampling and collecting platform (4), a mechanical hand multi-branch distribution system (5), and a software control system and a display interface of the workstation. The workstation is reasonable in structural design and high throughput and automation can be realized in a manual test so that a plurality of clinical samples can be treated and the working efficiency is improved; the aims of clinical applications and rapid screening of the samples are realized. The multifunctional workstation can be widely used for measuring cell outflow dynamic characteristics of other molecules, such proteins, lipids, RNA (Ribonucleic Acid), ions, saccharides, hormones, vitamins, and metabolites; the application range is wide.

Description

The multifunctional workstation that robotization high flux cholesterol detection and other analysis thing cell flow out

Technical field

The present invention relates to a kind of high throughput analysis equipment of biomolecule, be specifically related to a kind of activity that can detect HDL cellular cholesterol and flow out, also can be used for other molecules/compounds, quantitative as intraor extracellular streams such as protein, lipid, ion, hormone, metabolins, can be widely used in the multifunctional analysis system of high throughput automated biomolecule of biological study, clinical diagnosis and drug development.

Background technology

Angiocardiopathy is the chief threat of human health, has very high M & M, and occurs the trend that increases year by year, especially in the economy developed rapidly as China.According to the cardiovascular study on prevention center of the Ministry of Public Health, along with China's growth in the living standard and aging population, cardiovascular disease incidence rate every year with 30% speed increase.

Atherosclerotic causes cardiovascular disease incidence and main causes of death.The measure of at present conventional reduction hazards comprises and changes lifestyles and take lipid lowerers, but only can reduce by the coronary artery adverse events of about 30%, shows to need the new therapy of development badly to meet medical demand.Blood plasma HDL is based on it to angiocardiopathy, and particularly atherosclerotic natural protection capability, has become the important target in predicting cardiovascular class disease and treatment.But at present domestic and international market there is not yet similar provided by the invention can Efficient Evaluation HDL function screen the instrument of people at highest risk and evaluation result for the treatment of.In a word, the technical restriction of HDL Function detection has become the bottleneck of medicament research and development and clinical testing, and to develop new HDL diagnostic method and carry out functional analysis be present and even R&D direction in the quite a while, especially develop the high-precision detection technique of high flux.

It is relevant that anti-atherosis effect and it of HDL remove unnecessary cholesterol (i.e. Cholesterol Efflux) from arterial wall.The cholesterol being transported to liver is finally drained from ight soil, and this process is called reverse cholesterol transport.Present so-called cardiovascular health test is still based on the measurement of biomarker concentration, and the mark measured comprises plasma lipoprotein or the biomolecule relevant to inflammation and oxidative damage etc. can not meet current needs far away.Particularly its blood plasma level of function ratio of multinomial research display HDL is more important in recent years, and the latter is no longer considered to the reliability index of disease forecasting, and such as HDL can be caused its dysfunction by modifying.Clinical testing in recent years provides the activity of strong evidence display patient's blood plasma or HDL Cholesterol Efflux and arterial wall thickens and atherosclerotic remarkable improvement is positive correlativity, thus more can predict the generation of blood vessel and coronary artery class disease.

Cholesterol Efflux test is widely used in laboratory with the mechanism of study of disease, finds new drug candidate, and sets up and the associating of disease.Usually be applied to Cardiovascular Disease Study, but also comprise metabolism (as diabetes) and neurogenic disease etc.At present this experiment can only be undertaken by manual in laboratory, and manual operations relates to a large amount of diluted plasmas, cell cleaning and adds the work such as spline filter, and this experimental technique is time-consuming and easily have human error; In addition, repeated various cleaning and sample add the sample size that step not only limit each test, and a large amount of consumptive material, increase testing cost.Due to above defect, HDL functional test is very limited in the application aspect of clinical diagnosis or biotechnology/drug development, and cannot be used for the high flux screening of large sample needs.

Therefore, on the basis of existing technology necessary, a kind of automaticity of design research and development is high, can high flux, fast, accurately detects HDL cellular cholesterol and flows out the multifunctional analysis system that active and other biomolecule cell flows out.

Summary of the invention

Goal of the invention: the present invention is to solve the deficiencies in the prior art in HDL functional analysis, a kind of reasonable in design is provided, easy to operate, there is high flux, high precision, low cost, the advantages such as high-level efficiency, and the high throughput automated HDL functional analysis workstation had wide range of applications.

Technical scheme: in order to realize above object, the technical solution used in the present invention is:

The multifunctional workstation that a kind of robotization high flux cholesterol detection and other analysis thing cell flow out, it comprises constant temperature short incubation device, the cell be connected with constant temperature short incubation device to clean with application of sample platform, sample filtering platform, the sampling that is connected with sample filtering platform with collecting platform, cleans with cell the mechanical hand manifold delivery system be all connected with application of sample platform, sample filtering platform and sampling and collecting platform, the software control system of workstation and display interface.

Preferably, above-described high throughput automated multifunctional analysis workstation, described mechanical hand manifold delivery system comprises hole rectangular slab by all kinds of means, be arranged on the metal joint below hole rectangular slab by all kinds of means, the disposable sterilized rifle head be connected with metal joint, described disposable sterilized rifle head is by the control of automatically controlled mechanical hand along X-axis, and Y-axis and Z-direction move, thus can reach fast accurate from cell culture plate well and move the object of liquid.The present invention analyzes by utilizing the precise motion of automatically controlled mechanical hand to control cleaning step and collecting sample, and high flux repeats to realize the application of sample to Tissue Culture Plate multisample and cleaning by the automatic liquor-transferring system of central hyperchannel corresponding with 6 holes, 12 holes, 24 holes, 48 holes and 96 well culture plates.

Preferably, above-described high throughput automated multifunctional analysis workstation, simultaneously the function of described mechanical hand manifold delivery system is drawn the solution of tool precise volumes (0.01-1 milliliter), for flowing out serum-dilution in dynamic experiment and performing cleaning to cell and medicine/chemical treatment at particular point in time.

Preferably, above-described high throughput automated multifunctional analysis workstation, the temperature of described constant temperature short incubation device is 25 DEG C or 37 DEG C, is provided with controlled automatic opening and closing door, receivability 6 hole, 12 holes, 24 holes, 48 holes or 96 well culture plates.Be configured with sliding panel in described cell cleaning and application of sample platform, described sliding panel can insert in short-term constant-temperature incubator to be released cell cleaning and application of sample platform or is sent back to by culture plate in constant temperature short incubation device after task completes by culture plate.

Preferably, above-described high throughput automated multifunctional analysis workstation, described sample filtering platform base can place the porous filter plate had with culture plate identical standard size, utilizes gravity principle to filter and removes cell fragment and collect the adjustment nutrient culture media cultivating cell.

Described sampling and collecting platform are provided with multi-tube racks, and multi-tube racks is provided with collects test tube (bottle);

Described automatically controlled mechanical hand, after the porous filter plate collected specimens sample filtering platform, moves in the collection test tube (bottle) on sampling and collecting platform.

Multifunctional analysis system provided by the invention, wherein short incubation device can keep steady temperature (25 oc, 37 oc), the monolayer in Tissue Culture Plate is maintained; Cell cleaning and application of sample parts can carry out the high-efficiency washing of various intermediate steps needs, for (in advance) process cell and/or application of sample are prepared; Application of sample carries out in identical platform, and comprising can the biological sample of dilution process cell, as clinical serum, and by its simultaneously and rapidly application of sample in cultivation plate hole.The critical component of workstation is mechanical hand manifold delivery system, performs cleaning simultaneously, sample distributes, filters and collect multisample.At sample filtering platform, Cell and organism sample cultivate after collect nutrient culture media (containing 3h ] or fluorescently-labeled cholesterol) be filtered, then sampled by mechanical hand and move to and collect test tube (bottle).All above operations are all automatically switched by the setting of supporting software control system, and can programme voluntarily, thus can meet different experiments requirement, and workstation can automatically stored in information such as the numbering of a large amount of sample and analysis results.

Multifunctional analysis system provided by the invention is the technology platform of an open to the outside world.Except can the activity of the real time measure cholesterol efflux, also can measure cell flowing activity and its dynamics of other biomolecule, comprise protein, lipid, RNA, ion, carbohydrate, hormone, vitamin, metabolin etc.In addition, this workstation takes compatible strong global design, can dock with other platform or add-on module.Filtered fluid as above-mentioned collection can be sent to radic or fluorescence detector to quantize the concentration of labeled cholesterol; And the research that can be group class provides Sample pretreatment, application widely.

The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analysis thing cell flow out, have wide range of applications, as can be used as based on based on the experimental technique of cellular level diagnostic application instrument, comprise serum cholesterol and flow out active level, to assess the risk of angiocardiopathy.

The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analysis thing cell flow out also can be used as the charge service platform carrying out cell-based assay and diagnostic test, comprises the cell secreta using blood serum sample process cell and the blood serum sample described in recovery to supply subsequent analysis or detect in it.

The multifunctional workstation of a kind of robotization high flux cholesterol detection provided by the invention and other analysis thing cell outflow, also can be used as conveying and/or screening natural products, the synthesis adjustment outflow of compound and/or the instrument of emiocytosis effect in addition.As preferred further, workstation provided by the invention also can be used as the instrument of conveying and/or the outflow of screening cellular cholesterol.

In addition a kind of robotization high flux cholesterol detection provided by the invention and other analyze the multifunctional workstation that thing cell flows out, also can be used as qualification for regulating and/or the instrument of drug candidate of irritation cell Cholesterol Efflux.

The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analysis thing cell flow out, the effect of test-compound for various cell analysis thing can be screened, as lipid, protein, polypeptide, nucleic acid, ion, vitamin and/or enter intracellular exogenous factor as medicine, microbiotic, and/or pesticide.

Beneficial effect: the multifunctional analysis workstation of high throughput automated and other biomolecule provided by the present invention compared to the prior art tool has the following advantages:

Multifunctional analysis workstation provided by the present invention, reasonable in design, easy to operate, automaticity is high, and testing cost is low, has wide range of applications.

The principal feature of multifunctional analysis system is:

(1) high flux: can carry out synchro measure to a large amount of biology/clinical samples, thus eliminate manual retardation time and can save experimental period, manpower and reagent, testing cost is low;

(2) high precision: multifunctional analysis workstation provided by the invention, detection accuracy is high and reproducible, thus error between can avoiding criticizing, and to allow to compare better between different experiments room the/result of different experiments batch, and carries out analysis and the contrast of large data;

(3) versatility: multifunctional analysis workstation provided by the invention, can be connected with other automation equipment or instrument, can be used for cell and the much dissimilar experiment of system biology field, assist drug screening, the research of the aspect such as albumen and genomics, also can be widely used in hospital, clinic, company and research unit.

Accompanying drawing explanation

Fig. 1 is the structural representation of the multifunctional workstation that robotization high flux cholesterol detection of the present invention and other analysis thing cell flow out.

Fig. 2 is the structural representation of the mechanical hand manifold delivery system of multifunctional workstation of the present invention.

Embodiment

Below in conjunction with the drawings and specific embodiments, illustrate the present invention further, these embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention, after having read the present invention, the amendment of those skilled in the art to the various equivalent form of value of the present invention has all fallen within the application's claims limited range.

Embodiment 1

As depicted in figs. 1 and 2, the multifunctional workstation that a kind of robotization high flux cholesterol detection and other analysis thing cell flow out, it comprises constant temperature short incubation device (1), the cell be connected with constant temperature short incubation device (1) cleans and application of sample platform (2), sample filtering platform (3), the sampling be connected with sample filtering platform (3) and collecting platform (4), clean with cell and application of sample platform (2), the mechanical hand manifold delivery system (5) that sample filtering platform (3) and sampling are all connected with collecting platform (4), the software control system of workstation and display interface.

The multifunctional workstation that above-described robotization high flux cholesterol detection and other analysis thing cell flow out, described mechanical hand manifold delivery system (5) comprises hole rectangular slab (5-1) by all kinds of means, be arranged on the metal joint (5-2) below hole rectangular slab (5-1) by all kinds of means, the disposable sterilized rifle head (5-3) be connected with metal joint (5-2), described disposable sterilized rifle head (5-3) is controlled by automatically controlled mechanical hand, can along X-axis, Y-axis and Z-direction move, described metal joint (5-2) is connected with manifold system (5-4), described manifold system (5-4) top is provided with quantitative liquid-feeding pump (5-5), and manifold system (5-4) connects solution bottle (5-6), solution bottle (5-6) is provided with diverting valve (5-7), described solution bottle (5-6) is 5, five-way valve (5-8) is installed between manifold system (5-4) and solution bottle (5-6).

The liquid that moves of described disposable sterilized rifle head (5-3) amasss and is 0.01 to 1 milliliter, wherein the rifle head of 1 milliliter is used for carrying out the cleaning of sample, and the rifle head of 10 ~ 200 microlitres is used for accurately moving liquid to send blood plasma to be tried or other sample in the porous of Tissue Culture Plate.

The multifunctional workstation that above-described robotization high flux cholesterol detection and other analysis thing cell flow out, simultaneously the function of described mechanical hand manifold delivery system (5) is drawn the solution of 0.01 ~ 1 milliliter of precise volumes, for flowing out serum-dilution in dynamic experiment and performing cleaning to cell and medicine/chemical treatment at particular point in time.And described mechanical hand manifold delivery system (5) accurately can perform following functions: cell in plate hole is cultivated in cleaning, dilute serum, quick application of sample, filtered sample, collect the nutrient solution for analyzing.

The multifunctional workstation that the above robotization high flux cholesterol detection and other analysis thing cell flow out, the temperature of described constant temperature short incubation device (1) is 25 DEG C or 37 DEG C, be provided with controlled automatic opening and closing door, receivability 6 hole, 12 holes, 24 holes, 48 holes or 96 well culture plates;

And be configured with sliding panel in described cell cleaning and application of sample platform (2), described sliding panel can insert in short-term constant-temperature incubator (1) to be released cell cleaning and application of sample platform (2) or is sent back to by culture plate in constant temperature short incubation device (1) after task completes by culture plate.

The multifunctional workstation that above-described a kind of robotization high flux cholesterol detection and other analysis thing cell flow out, the base of described sample filtering platform (3) is provided with the porous filter plate with culture plate identical standard size;

Described sampling and collecting platform (4) are provided with multi-tube racks, and multi-tube racks is provided with collection test tube;

Described automatically controlled mechanical hand (5) after the porous filter plate collected specimens sample filtering platform (3), move to sampling and collecting platform (4) on collection in vitro.

The multifunctional workstation that robotization high flux cholesterol detection provided by the invention and other analysis thing cell flow out, because of its quick and high-throughout feature, the range of application of platform comprises:

1) automated analysis Cholesterol Efflux

A. for the research of fundamental research-atherosclerotic, lipid and lipoprotein metabolism, HDL function.Platform provides desirable laboratory facilities, to promote biomedical research and drug development by for the detection method for the purpose of dynamics and research.

B. screen and clinical detection for medicament research and development and clinical detection-high flux lead compound.

2) cell of automated analysis various biomolecules flows out

For measuring intraor extracellular stream and the dynamics thereof of other biomolecule, comprise protein, lipid, RNA, ion, carbohydrate, hormone, vitamin, metabolin etc.

3) platform is cultivated/dealt with the work to Universal Cell

Can be connected with many biological experiment operating systems and carry out dissimilar cell and systems biology is tested, as the research of protein science and genomics aspect.

The basic mechanical design feature index of the multifunctional workstation instrument that robotization high flux cholesterol detection provided by the invention and other analysis thing cell flow out :

Each sample size: 1-96(can many plates)

Move liquid flux: 0.01-1 ml, high precision moves liquid head (error +2%)

Measuring Time: (0,5,10 minutes to 10 hours)

Measuring tempeature: 25 oC, 37 oc

Systems Sterilizer: ultraviolet

Power supply: 220/110 AC; 50-60HZ

Operating temperature: 18-37 oC

Multifunctional analysis system instrument provided by the invention, can detect a large amount of biology/clinical samples simultaneously, and detection time is a few minutes arrive several hours, and workstation operation is accurate and repeatability is high, thus can reduce/eliminate personal error, improves the accuracy detected.

Embodiment 2

Now with clinical detection HDL Cholesterol Efflux for embodiment, the principle of work of this multifunctional workstation and concrete operation steps process are described.The mammalian macrophage (as J774 cell) that test cell line can adopt laboratory usually to apply, include [ 3h] or other tracer agent, as fluorescently-labeled cholesterol.Cell can carry out pre-service with biochemical reagents usually, as passed through cAMP active cell Membrane cholesterol transporter ABCA1.More than mark is relative with the process operation of active cell simple and easy, not within the scope of robotization of the present invention.

Test cell line is inoculated in as required in 6,12,24,48 or 96 well culture plates, grow in serum free medium, and allow it be exposed in the air of constant temperature short incubation device (1), nutrient culture media contains stable buffer solution, as HEPES or other damping fluid, cell can from 0 to 10 hours not etc., and can spend the night or hatching in 24 hours as required cultivating in plate hole time of growing.

The sliding panel that when experiment starts, cleaning and application of sample platform (2) configure inserts short incubation device (1), experimental plates is moved out to cleaning and application of sample platform (2), move liquid head by 1 ml of automatically controlled mechanical hand, rinse the cell in cultivation plate hole with Hank balanced salt solution and repeat twice; Then bovine serum albumin(BSA) (BSA) solution placing adding 0.2% in hole is used as to close for 1 hour, to reduce background signal; Then cell in culture plate (6) hole is rinsed three times with serum free medium.The quantity of aseptic rifle head (5-3) that is connected of hole rectangular slab (5-1) will by functional unit software control by all kinds of means with mechanical hand, keeps and the hole count of culture plate matches.

At mechanical hand manifold platform, move liquid head to automatically switch under software the aseptic rifle head (5-3) varied in size, clinical serum sample the to be tried nutrient culture media containing HEPES damping fluid is carried out being diluted to variable concentrations as 10%, 5%, 2% and 1%.

Diluted plasma sample is divided deliver to cleaning and application of sample platform (2) Tissue Culture Plate (6) hole in, and by sliding panel, culture plate (6) is sent into constant temperature short incubation device (1), close chamber's door shares and hatches 4 hours (37 oc).If experiment purpose is dynamics (time dependence) research, need at different time points, as culture plate to take out and to draw the supernatant nutrient solution in respective aperture by 0,1,2,4 and 8 hour, move to the porous filter plate on filter stage (3).

After cultivation completes, culture plate is put back to cleaning and application of sample platform (2), draw the supernatant nutrient solution in every hole by aseptic rifle head (5-3) and move to the porous filter plate on filter stage (3), porous filter plate has the standard size identical with culture plate and hole count, utilizes gravity principle to filter and removes cell fragment possible in supernatant.

Automatically controlled mechanical hand is from the quantitative filtration nutrient culture media of the porous filter plate collection filter stage (3), then move to and collecting platform (4) is placed on microcentrifugal tube on pipe support or other collects in test tube (bottle), for the concentration in order to quantize labeled cholesterol of radiological measuring or fluorescence detector.This concentration value is used for calculating the Cholesterol Efflux rate of macrophage.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (9)

1. the multifunctional workstation of a robotization high flux cholesterol detection and other analysis thing cell outflow, it is characterized in that, it comprises constant temperature short incubation device (1), the cell be connected with constant temperature short incubation device (1) cleans and application of sample platform (2), sample filtering platform (3), the sampling be connected with sample filtering platform (3) and collecting platform (4), clean with cell and application of sample platform (2), the mechanical hand manifold delivery system (5) that sample filtering platform (3) and sampling are all connected with collecting platform (4), the software control system of workstation and display interface,
Described mechanical hand manifold delivery system (5) comprises hole rectangular slab (5-1) by all kinds of means, be arranged on the metal joint (5-2) below hole rectangular slab (5-1) by all kinds of means, the disposable sterilized rifle head (5-3) be connected with metal joint (5-2), the position of described disposable sterilized rifle head (5-3) is controlled by automatically controlled mechanical hand, can along X-axis, Y-axis and Z-direction move, described metal joint (5-2) is connected with manifold system (5-4), described manifold system (5-4) top is provided with quantitative liquid-feeding pump (5-5), and manifold system (5-4) connects solution bottle (5-6), solution bottle (5-6) is provided with diverting valve (5-7), described solution bottle (5-6) is 5, five-way valve (5-8) is installed between manifold system (5-4) and solution bottle (5-6),
Simultaneously the function of described mechanical hand manifold delivery system (5) is drawn the solution of 0.01 ~ 1 milliliter of precise volumes, for flowing out serum-dilution in dynamic experiment and performing cleaning to cell and medicine/chemical treatment at particular point in time.
2. the multifunctional workstation of robotization high flux cholesterol detection according to claim 1 and other analysis thing cell outflow, it is characterized in that, mechanical hand manifold delivery system (5) accurately can perform following functions: cell in cleaning culture plate (6) hole, dilute serum or other wait try biological sample, quick application of sample, filtered sample, collects the nutrient solution for analyzing.
3. the multifunctional workstation of robotization high flux cholesterol detection and other analysis thing cell outflow according to claim 1, it is characterized in that, the temperature of described constant temperature short incubation device (1) is 25 DEG C or 37 DEG C, be provided with controlled automatic opening and closing door, receivability 6 hole, 12 holes, 24 holes, 48 holes or 96 well culture plates;
Be configured with sliding panel in described cell cleaning and application of sample platform (2), described sliding panel can insert in short-term constant-temperature incubator (1) to be released cell cleaning and application of sample platform (2) or is sent back to by culture plate in constant temperature short incubation device (1) after task completes by culture plate.
4. the multifunctional workstation of robotization high flux cholesterol detection according to claim 1 and other analysis thing cell outflow, it is characterized in that, the base of described sample filtering platform (3) is provided with the porous filter plate with culture plate identical standard size;
Described sampling and collecting platform (4) are provided with multi-tube racks, and multi-tube racks is provided with collection test tube; Described mechanical hand manifold delivery system (5) after the porous filter plate collected specimens sample filtering platform (3), move to sampling and collecting platform (4) on collection in vitro.
5. the robotization high flux cholesterol detection described in any one of claim 1-4 and other analyze multifunctional workstation that thing cell flows out as based on based on the experimental technique of cellular level diagnostic application instrument, comprise serum cholesterol and flow out active level, to assess the risk of angiocardiopathy.
6. the multifunctional workstation that the robotization high flux cholesterol detection described in any one of claim 1-4 and other analysis thing cell flow out, as the charge service platform carrying out cell-based assay and diagnostic test, comprises the cell secreta that the serum/media samples after using blood serum sample process cell and the process described in recovery supplies subsequent analysis or detects in it.
7. the robotization high flux cholesterol detection described in any one of claim 1-4 and other multifunctional workstation analyzing the outflow of thing cell are as conveying and/or screening natural products, the synthesis adjustment outflow of compound and/or the instrument of emiocytosis effect.
8. the instrument that the robotization high flux cholesterol detection described in any one of claim 1-4 and other multifunctional workstation analyzing the outflow of thing cell flow out as conveying and/or screening cellular cholesterol.
9. the robotization high flux cholesterol detection described in any one of claim 1-4 and other analyze multifunctional workstation that thing cell flows out as qualification for regulating and/or the instrument of drug candidate of irritation cell Cholesterol Efflux.
CN201310702939.8A 2013-12-19 2013-12-19 Multifunctional workstation for automatically detecting outflow of cholesterol and other analyte cells with high throughput CN103645332B (en)

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