DE10053747C2 - Method for the simultaneous determination of two fluorescence emissions with a single laser flow cytometer - Google Patents

Description

The present invention relates to a method for simultaneous determination of the fluorescence emissions of a first fluorochrome and a second fluorochrome, which an emission overlapping with the first fluorochrome has a wavelength range.

Flow cytometry is a procedure that helps the Fluorescence of single cells marked with a fluorochrome can be measured. A flow cytometer includes one Storage container with a cell suspension of marked cells, a falling distance that is a thin droplet of droplet that is made of flows out of an opening of the storage container, cross must, a laser that irradiates the droplet thread, and one or more measuring channels, which the Can measure emission fluorescence of the droplet filament. The Production of the droplet thread is critical here the aim is to form individual droplets that each contain only one cell. That way can be achieved that the laser also only one cell at a time irradiated so that the fluorescence of the measuring channels single cell is measured. An attached Data processing system stores the individual measurements and can carry out a statistical evaluation.

DE 42 10 970 C2 describes a method for simultaneous Determination of the fluorescence emissions of a first Fluorochrome and a second fluorochrome, which one with the first fluorochrome overlapping emission waves has length range.

US 61 00 038 A describes a method for simultaneous Determination of the fluorescence emissions of a first Fluorochrome and a second fluorochrome in one Flow cytometer with two fluorescence channels different reception wave ranges.

A principle of flow cytometry is the measurement of relative fluorescence using voltage-controlled photomultiplier tubes (PMTs) as measuring channels. The fluorescence signal is displayed on a logarithmic scale from 10 ⁰ (beginning of the 1st decade) to 10 ⁴ (end of the 4th decade). A calibration to this range of values takes place on the basis of negative and positive control standards, which in one case should only have background fluorescence and in the other case the maximum expected fluorescence to be determined.

The PMT voltages used are from the used Flow cytometer dependent and are based on the respective control standards used.

In addition, electronic compensation of the measured values be made. Electronic compensation is on Standard procedure in multicolor flow cytometry. Become Cells marked with two or more fluorochromes are formed sometimes the problem that due to the overlap of the Emission fluorescence spectra with strong fluorescence signals radiation into the neighboring fluorescence channel occurs and there leads to a false positive signal. This is done in the context of electronic compensation electronic subtraction for each individual measurement event corrected by the false positive fluorescence Percentage of the starting fluorescence is subtracted.

The problem in flow cytometry sometimes arises that you have two different fluorochromes at the same time characterize different physiological characteristics want to measure, their emission peak wavelengths like this are close together that the emissions, spread over the usual wavelength range, overlap. A simultaneous measurement of such fluorochromes has been involved a single laser flow cytometer even when used Electronic compensation is not possible, as at least often in one of the fluorescence channels an overlay of the for the other fluorescence channel certain signal took place so that the signal to be measured in this fluorescence channel is not that fluorochrome measured with it reflected. At this Situation had to be on multi-laser flow cytometer, for example using UV-stimulable substrates and a UV excitation. These devices are very expensive and complex to use. With the usual ones clinical laboratories and research facilities  A laser routine device is the simultaneous measurement of such neighboring emitting fluorochromes were not possible. An example of such a situation in which certain Fluorochromes must be used to get the one you want To be able to make statements about the cells examined in the field of apoptosis examination.

Apoptosis is a biological process in which a chain of genetically controlled events are triggered in cells the controlled death and the efficient elimination of the affected cells.

Detection of apoptosis and apoptosis-regulating molecules is crucial for diagnosis and therapy of various Diseases such as autoimmune diseases, viral infections, neurodegenerative diseases, bone marrow insufficiency syndromes, leukaemias and solid tumors.

One of the possible applications is cytostatics testing. Cytostatics induce apoptosis by doing essential Activate elements of the apoptosis program. The effectiveness of cytostatics depends on the extent to which molecular apoptosis regulators can be activated.

This is an important factor in determining apoptosis Level of caspase activation. The Caspase Protein Family causes protein breakdown in apoptosis. A flow cytometric determination of caspase activation possible through the use of special rhodamine 110 derivatives. Rhodamine 110 is a fluorochrome Wavelength of 497 nm can be optimally excited and then produces a fluorescence emission, the peak of which is at 520 nm lies. The fluorochrome rhodamine 110 is made with peptides coupled, which during a caspase activation of these Caspases are cleaved from the Rhodamine 110 again. Through the The fluorochrome property of Rhodamine 110 is eliminated activated and can be measured in the flow cytometer.  

Another characteristic of apoptosis is that Mitochondrial activation with a decrease in Mitochondrial membrane potential. Through the Use of mitochondrial membrane potential sensitive Fluorochrome is also determining these changes in the Mitochondrial membrane potential of a flow cytometric Examination accessible. An example that can be used Substance is 3,3'-dihexyloxacarbocyanine iodide (DiOC6 (3)). This Substance is optimal at a wavelength of 484 nm excitable. Both Rhodamine 110 and DiOC6 (3) can use it practically with a laser, for example an argon laser from 488 nm wavelength can be excited. However, the peak is the fluorescence emission of DiOC6 (3) at 501 nm, and thus only 19 nm from the peak of Rhodamine 110. from that the problem arises that with a normal measurement of the Fluorescence emission the Rhodamine 110 fluorescence emission an accurate measurement of the DiOC6 (3) measurement on another Fluorescence channel prevented.

The present invention is based on the object To provide methods by means of which even with simple Single laser flow cytometers measure two of these initially colliding fluorochrome becomes possible.

This object is achieved by the method according to the independent claim 1.

Further advantageous configurations, aspects and details result from the dependent claims, the Description and the attached figure.

The invention is based on the surprising finding that one by lowering the photomultiplier tube voltage the fluorescence channel on which the first fluorochrome to be measured, the signal of the disturbing second Can suppress fluorochrome so far that a flawless Measurement of the affected first fluorochrome is possible.  

In particular, the invention is based on the finding that Mitochondrial membrane-sensitive fluorochrome can be measured simultaneously with Rhodamine 110 if on Fluorescence channel for measuring the Mitochondrial membrane-sensitive fluorochrome Photomultiplier tube voltage compared to normal is reduced and thereby a Rhodamin 110 interference signal can be sufficiently suppressed.

The invention is therefore directed to a method for the simultaneous determination of the fluorescence emissions of a first fluorochrome and a second fluorochrome, which has an emission wavelength range overlapping with the first fluorochrome, in a single-laser flow cytometer with two fluorescence channels of different reception wave ranges, the method comprising does the following:

• A) Excitation of the first fluorochrome with this Fluorochrome labeled reference cells with a for both fluorochromes suitable laser beam;
• B) Setting the photomultiplier tube voltage of the second fluorescence channel, which is connected to the emission of the second fluorochrome is adapted to one Value at which none of the first fluorochrome in the second Fluorescence channel generated Fluorescence emission signal is detected;
• C) Exciting the Flurochrome from with the first and second Fluorochrome marked cells to be measured with the Laser beam;
• D) Measuring the fluorescence emission signal of the first Fluorochrome in the cells to be measured in the first Fluorescence channel, which is associated with the emission of the first Fluorochrome is adjusted

and

• A) Measuring the fluorescence emission signal of the second Fluorochrome in the cells to be measured in the second Fluorescence channel.

Excitation is understood here to mean that the laser beam is directed at the droplets with the cells and thereby it emission fluorescence occurs in or on the cells. This fluorescence is measured using the fluorescence channels, the spectral sensitivity of these to expected emission must be adjusted to the fluorescence to be able to perceive. The fluorescence generated is considered a Signal referred to, which in the fluorescence channels can be quantified (measured).

The method according to the invention makes use of a Calibration method using reference cells. These are defined as those cells in their Properties as far as possible of the cells to be determined correspond, but which are in a "zero state", at which has not been manipulated with the cells which lead to a change or formation of fluorescence. In contrast, the cells to be measured are those in which the processes to be investigated have been triggered or carried out and which therefore have an altered fluorescence.

The method according to the invention can thereby further in its Measurement accuracy can be improved that different, further Calibration and control steps are introduced. So it can The procedure may be changed in that on the second Fluorescence channel additionally electronic compensation of 60-90%. The electrical compensation on second fluorescence channel can, for example, advantageously 80 %. This measure allows the invention desired suppression of cross signals further improved become.  

The following steps can also be carried out before step A:

• - Excitation of unmarked reference cells with the Laser beam; and
• - Setting the generated by the fluorescence channels Signals to a medium value within the first decade of a measurement scale.

These are standard calibration measures because a flow cytometer, as explained above, relative values certainly.

It is also possible that the method according to the invention has the following additional steps before step C:

• - Excitation of the second fluorochrome with this Fluorochrome labeled reference cells with the Laser beam;
• - Setting an electronic compensation on the first fluorescence channel, so that no signal is measured above the first decade (10 ¹ ) on the first fluorescence channel;
• - save the setting;

and that the following further control steps after step D can have:

• - Excitation of the second fluorochrome with this Fluorochrome marked cells to be measured with the Laser beam;
• - Setting the saved setting;
• - Measuring the fluorescence emission signal of the second  Fluorochrome in the cells to be measured in the second Fluorescence channel; and
• - Compare the measured signal with that in Step D measured signal.

This measure checks whether a correct signal with respect to the second fluorochrome can be measured in the second channel. The compensation set ensures that the measured fluorescence values are between 10 ⁰ and 10 ¹ , ie between 1 and 10 on the measuring scale.

As an additional control, the method can have the following further control steps:

• - Excitation of the first fluorochrome with this Fluorochrome marked cells to be measured with the Laser beam;
• - Measure the fluorescence emission signal of the first Fluorochrome in the cells to be measured in the first Fluorescence channel;
• - Compare the measured signal with the stored signal and with the during measurement in Step D signal obtained.

This additional control advantageously requires that the storage of the measured value outlined above takes place is.

With these procedural steps and their combinations an accurate, controlled measurement of two fluorochromes in perform a one-laser flow cytometer, which makes Get rid of previous methodological difficulties.

For example, the methodological difficulty of simultaneous measurement of Rhodamine 110 and  potential-sensitive fluorochromes in the overlap of the Fluorescence spectra that are not in the prior art allows to measure both fluorochromes at the same time. rhodamine 110 has a strong emission at 540 nm, the wavelength at which the potential-sensitive fluorochromes are measured. The means that a positive rhodamine 110 signal in the first Fluorescence channel (e.g. channel FL2 at Becton-Dickinson flow meters) an additional signal in the second fluorescence channel (FL1) of the flow cytometer causes. Reduce the photomultiplier tube voltage FL1 also increases the sensitivity to the radiation of Rhodamine 110 from the first to the second fluorescence channel reduced.

After reducing the photomultiplier tube voltage, it succeeds the radiation into the FL1 channel (second channel) prevent. For measuring the mitochondrial DiOC6 (3) is the reduction of membrane potential Photomultiplier tube voltage harmless, as a normal one Membrane potential is an extremely strong fluorescence signal produced and even at reduced Photomultiplier tube voltage can be measured. Also the Mitochondrial membrane potential can be lowered despite Reliable reduction of the photomultiplier tube voltage can be detected.

If the present invention for the Rhodamine 110 / DiOC6 (3) Fluorochrome pair should be used, the Peptide-Rhodamine 110 substrate concentration and the DiOC6 (3) concentration for the application of the invention Method kept in a narrow area and adhered to the Flow cytometer settings can be adjusted. The Fluorescence of the released rhodamine 110 and Membrane potential sensitive fluorochrome can then simultaneously at single cell level using single laser flow cytometry be quantified.

Therefore, the method is preferably for simultaneous  fluorometric determination of at least two Apoptosis parameters used.

Here, as shown, the selected parameters activation of caspases and activation of mitochondria his. Caspase activation is preferably by measurement the degradation of a caspase target peptide determined during mitochrondrial activation by measuring Changes in membrane potential, especially of reductions the mitochrondrial membrane can be determined.

According to the intended use, the first fluorochrome can at least be a caspase-cleavable rhodamine 110 derivative. For example, it may have a peptide rhodamine 110.

Since there is a whole range of caspases in cells, leaves through the specific adaptation of the rhodamine derivatives Behavior of a specific one for this rhodamine derivative find out specific caspase.

It is also possible to add more than one rhodamine 110 derivative use. Typical caspase-specific, preferred rhodamine 110 derivatives are (AspGluValAsp) 2-rhodamine 110 [= (DEVD) 2- Rhodamine 110] and / or (Asp) 2-Rhodamine 110.

The second fluorochrome may preferably be one Membrane potential sensitive fluorochrome, for example 3,3'-dihexyloxacarbocyanine iodide.

Depending on the fluorochrome used, there will be the excitation mediated by the laser beam in its Distinguish wavelength. In the preferred setting for testing the apoptosis behavior of cells is stimulated preferably at a wavelength of 488 nm, for example using an argon laser.

In a specific embodiment, by application of substrates that specifically the activity of individual  Detect caspase isoenzymes, the position of individual Caspaseniosoenzyme in relation to the activation of the Mitochondria are identified (initiator and Effector caspase). For the further development of cytostatics is it is crucial to identify substances that are both Activate signal paths, because only then from a safe Apoptosis induction can be assumed. Through the application Flow cytometry is the method according to the invention especially for testing chemosensitivity primary Tumor and leukemia cells are suitable because they are in them heterogeneous cell populations potentially resistant Subpopulations can be identified.

Furthermore, the method according to the invention permits Connection of the activity measurement of two signal paths with the Detection of surface epitopes using fluorochromated Antibody. This makes it possible to be resistant and sensitive Subpopulations continue, for example regarding their Characterize degree of differentiation. Finally enables the inventive method through the connection with the detection of phosphatidylserine externalization a comprehensive monitoring of the early ones using Annexin-V Apoptosis steps of caspase and mitochondrial activation up to a late step.

The method according to the invention is applicable to a number of Areas of application can be used that are about the clarification of Go beyond apoptosis signal paths and generally independent Make events in cells correlated with each other. That the Principle underlying the invention is not of the Use of rhodamine and membrane potential sensitive Fluorochrome dependent but can be in any Problems with similar fluorochrome requirements be used. The decisive advantage of The inventive method is the low cost, a simple and quick feasibility, as well as the Possibility of currently available routine flow cytometers for to use the measurement. This ensures that  Method according to the invention as a diagnostic application for Chemosensitivity measurement in leukaemias and tumors, for example, will spread rapidly.

The invention is further explained with reference to the figures, which demonstrate:

Fig. 1a: Jurkat cells without cytarabine treatment;

FIG. 1b: Jurkat cells with a three-hour cytarabine treatment; and

Fig. 1c: Jurkat cells with a six-hour cytarabine treatment. The contour plot of FIG. 1 in this case shows the DiOC6 (3) signal in the FL1 versus Rhodamine 110 signal in FL2.

The invention is illustrated below using an example be, with reference to the accompanying figure becomes.

example

In this exemplary embodiment, Jurkat cells and primary leukemia cells are used, which are in a RPMI 1640 buffer with 10% heat-inactivated fetal calf serum, 200 mM glutamine, and 100 U / ml penicillin / streptomycin in a concentration of 0.5 to 1 , 0 × 10 ⁶ cells / ml. Rhodamine 110 (R) -labeled peptides of the structure (Asp) 2-Rhodamine 110 (D2R) and (Z-DEVD) 2R with an additional N-benzoylcarbonyl group on the N-terminal Asp residue, were synthesized, the coupled peptides had a purity of at least 95%. The stock solution is a DSMO-ethanol mixture in a ratio of 1: 1.

The CD-95 receptor stimulation, which is used in the present example of the invention, is carried out by means of the monoclonal antibody anti-APO-1 (3) at a concentration of 1 µg / ml in the presence of 10 ng / ml Protein A. performed as a crosslinker, the apoptosis being carried out in 1 ml (at 10 ⁶ cells / ml) on cells. The cells are washed in PBS and 2-mercaptoethanol is added to a final concentration of 10 mM.

The cells pretreated in this way can now be treated with the Fluorochromes are examined. For this, the Rhodamine-labeled peptide substrates in a final concentration of typically 50 microns of the cell suspension added. To Washing in 1 × PBS and resuspension in 300 μl 1 × PBS can this approach will be subjected to a FACS analysis to as To serve control.

In order to be able to measure mitochrondrial membrane potential changes and rhodamine derivative cleavage, cells are resuspended with PBS-FACS in a concentration of 5 × 10 ⁵ cells / ml and with for example D2R and DiOC6 (3) at a concentration of 460 ng / ml for incubated for twelve minutes at 37 ° in the dark. This is immediately followed by an analysis in the flow cytometer. As a control for downregulated mitochondrial membrane potential differences, the cells are also incubated with carbonylcyanide-m-chlorophenylhydrazone (mCICCP).

The following control standards are introduced:

• 1. With unlabelled cells, ie without and without Rhodamine 110 DiOC6 (3), the signal appears in all fluorescence channels an average value within the first decade of logarithmic measuring scale of the cytometer set (Autofluorescence Standard).
• 2. Cells labeled with DiOC6 (3) that have not been incubated with rhodamine 110, the electronic compensation for the first fluorescence channel, for example FL2 in Becton-Dickenson devices, is set so that no fluorescence above 10 ^{1 is} measured in FL2 (compensation standard for the first fluorescence). This device setting is saved for step 4.
• 3. cells labeled with rhodamine 110, in which apoptosis was induced, which is a positive caspase signal in the two fluorescence channels used, the Fluorescence in the second channel, for example FL1, compensated. To do this, the electronic compensation is on Values set by 80% and the Photomultiplier tube voltage as reinforcement for the second channel reduced so far that no Fluorescence signal is detected in the second channel (Compensation standard for the first fluorochrome, Rhodamine 110).
• 4. DiOC6 (3) labeled cells that are not rhodamine 110 are marked and in which apoptosis is triggered has now been checked, whether a correct one DiOC6 (3) signal measured on the second fluorescence channel FL1 can be (standard for DiOC6 (3)). To do this, the Device settings, which are saved under 2. were restored for a short time and that recorded signal with the signal of the device setting compared under 3.
• 5. Rhodamine 110 is labeled as an additional control Cells in which apoptosis was induced with the Device settings under 2. and the final Settings measured under 3. and the signals compared.

In addition, in the method according to the invention, the concentration of DiOC6 (3) can be adjusted so that with a conventional flow cytometer setting (according to the autofluorescence standard a normal mitochondrial membrane potential) a signal in the fourth decade (10 ³ -10 ⁴ ) in the second fluorescence channel, in The present example FL1 is generated while an apoptosis-related reduced membrane potential is measured in the third decade. A mitochondrial membrane potential reduction can thus be reliably measured even with a considerably reduced photomultiplier tube voltage, which is required for the compensation of the Rhodamine 110 signal.

Since rhodamine 110 has an emission at 540 nm, it produces an Signal in fluorescence channel 1 (FL1) by also DiOC6 (3) signal is measured. To solve this problem will eliminate the FL1 photomultiplier tube voltage decreased to increase sensitivity to the Rhodamine 110 signal reduce. After lowering the photomultiplier tube voltage, it is possible to compensate for the Rhodamine 110 signal and thus it to be completely eliminated from the FL1 channel.

Detection of a degraded Mitochondrial membrane potential is reduced by reducing the Photomultiplier tube voltage is not affected as that Mitochondrial membrane potential a strong DiOC6 (3) signal generated, even with reduced Photomultiplier tube voltage can be detected.

The analyzes shown in Fig. 1 were carried out with a commercially available cytometer equipped with a 488 nm argon laser and a 650 nm red diode laser. At least 50,000 events per sample were recorded, saved in list mode files and then analyzed with the associated software.

Detection of the D2R cleavage can be done by measuring the Mitochondrial membrane potential can be combined. For this become Jurkat cells with 10 µg cytarabine for three or treated for six hours. The cells will incubated simultaneously with D2R and DiOC6 (3).  

The inventive method thus allows simultaneous Measurement of two independent fluorochrome signals using conventional flow cytometry in living Cells.

Claims (16)

1. A method for the simultaneous determination of the fluorescence emissions of a first fluorochrome and a second fluorochrome which has an emission wavelength range overlapping with the first fluorochrome, in a single-laser flow cytometer with two fluorescence channels of different reception wavelength ranges, the method comprising the following steps:

• A) exciting the first fluorochrome of reference cells labeled with this fluorochrome with a laser beam suitable for both fluorochromes;
• B) adjusting the photomultiplier tube voltage of the second fluorescence channel, which is adapted to the emission of the second fluorochrome, to a value at which no fluorescence emission signal generated by the first fluorochrome in the second fluorescence channel is detected;
• C) exciting the flurochromes of cells to be measured marked with the first and second fluorochrome with the laser beam;
• D) measuring the fluorescence emission signal of the first fluorochrome in the cells to be measured in the first fluorescence channel, which is adapted to the emission of the first fluorochrome;

and

• A) Measuring the fluorescence emission signal of the second fluorochrome in the cells to be measured in the second fluorescence channel.

2. The method according to claim 1, characterized in that on second fluorescence channel additionally an electronic one Compensation of 60-90% is made. 3. The method according to claim 2, characterized in that electrical compensation on the second fluorescence channel Is 80%. 4. The method according to any one of claims 1 to 3, characterized in that the following steps are carried out before step A:

• - excitation of unmarked reference cells with the laser beam; and
• - Setting the signal generated by the fluorescence channels to an average value within the first decade of a measurement scale.

5. The method according to any one of claims 1 to 4, characterized in that it has the following additional steps before step C:

• - Excitation of the second fluorochrome from reference cells marked with this fluorochrome with the laser beam;
• - Setting an electronic compensation on the first fluorescence channel so that no signal is measured above the first decade (101) on the first fluorescence channel;
• - save the setting;

and that it has the following further control steps after step D:

• - Excitation of the second fluorochrome of cells to be measured marked with this fluorochrome with the laser beam;
• - Setting the saved setting;
• - Measuring the fluorescence emission signal of the second fluorochrome in the cells to be measured in the second fluorescence channel; and
• - Compare the measured signal with the signal measured in step D.

6. The method according to claim 5, characterized in that it has the following further control steps:

• - Excitation of the first fluorochrome of cells to be measured marked with this fluorochrome with the laser beam;
• - Measuring the fluorescence emission signal of the first fluorochrome in the cells to be measured in the first fluorescence channel;
• - Compare the measured signal with the stored signal and with the signal obtained in the measurement in step D.

7. The method according to any one of claims 1 to 6, characterized characterized that it is for simultaneous fluorometric determination of at least two Apoptosis parameters is used. 8. The method according to claim 7, characterized in that the parameters the caspase activation and the Mitochondrial activation are. 9. The method according to claim 8, characterized in that the activation of caspases by measuring the degradation  of a caspase target peptide is determined. 10. The method according to claim 8 or 9, characterized in that mitochondrial activation by measuring Changes in the membrane potential of the mitochondrial membrane is determined. 11. The method according to any one of claims 1 to 10, characterized characterized in that the first fluorochrome at least one Caspase-cleavable Rhodmin 110 derivative. 12. The method according to claim 11, characterized in that the rhodamine 110 derivative is a peptide rhodamine 110 having. 13. The method according to claim 11 or 12, characterized characterized in that the rhodamine 110 derivative (AspGluValAsp) 2-rhodamine 110 and / or (Asp) 2-rhodamine 110 contains. 14. The method according to any one of claims 1 to 13, characterized characterized in that the second fluorochrome one Is membrane potential sensitive fluorochrome. 15. The method according to claim 14, characterized in that the membrane potential-sensitive fluorochrome 3,3'-dihexyloxacarbocyanine iodide. 16. The method according to any one of claims 1 to 15, characterized characterized that the excitation at one wavelength of 488 nm.