Method of analysing a cell or other biological material containing a nucleic acid
11255761 · 2022-02-22
Assignee
Inventors
- Paul James Smith (Vale of Glamorgan, GB)
- Rachel J. Errington (Leicestershire, GB)
- Laurence Hylton Patterson (Leicestershire, GB)
Cpc classification
G01N1/30
PHYSICS
C09B1/207
CHEMISTRY; METALLURGY
International classification
G01N1/30
PHYSICS
C09B1/20
CHEMISTRY; METALLURGY
Abstract
According to the invention there is provided a compound of Formula (I) in which: A is a C.sub.2-8 alkylene group; R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from hydrogen, C.sub.1-4 alkyl, C.sub.2-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, or R.sup.2 and R.sup.3 together form a C.sub.2-6 alkylene group which with the nitrogen atom to which R.sup.2 and R.sup.3 are attached forms a heterocyclic ring; X.sub.1, X.sub.2 and X.sub.3 are independently selected from hydrogen, hydroxyl, NR.sup.1-A-NR.sup.2R.sup.3R.sup.4+(Z.sup.m−).sub.1/m, for halogeno amino, C.sub.1-4 alkoxy or C.sub.2-8 alkanoyloxy; and (Z.sup.m−).sub.1/m is an anion of charge m; or a derivative in which the group NR.sup.1 is quaternarized.
Claims
1. A composition suitable for use as a fluorescent dye for analyzing cells or other biological materials, comprising a mixture of: a fluorescent, cell-impermeant, compound of Formula (I) ##STR00008## or a derivative in which the group NR' is quaternarized, wherein A is a C.sub.2-8 alkylene group; R.sup.1 selected from hydrogen, C.sub.1-4 alkyl, and C.sub.3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, and R.sup.2, R.sup.3, and R.sup.4 are independently selected from C.sub.1-4 alkyl, and C.sub.3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, or R.sup.1 is selected from hydrogen, C.sub.1-4 alkyl, and C.sub.3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, R.sup.4 is selected from C.sub.1-4 alkyl, and C.sub.3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, and R.sup.2 and R.sup.3 together with the nitrogen atom to which R.sup.2 and R.sup.3 are attached form a heterocyclic ring, wherein the heterocyclic ring consists of a C.sub.24 alkylene group together with the nitrogen atom; X.sub.1, X.sub.2, and X.sub.3 are independently selected from hydrogen, hydroxyl, NR.sup.1-A-NR.sup.2R.sup.3R.sup.4+(Z.sup.m−) .sub.1/m, halogeno amino, C.sub.1-4 alkoxy, and C.sub.2-8 alkanoyloxy; and (Z.sup.m−) .sub.1/m is an anion of charge m, where m is 1, 2 or 3; together with at least a second fluorochrome or light-emitting compound, wherein the compound is cell permeant and is of Formula (II) ##STR00009## or an N-oxide thereof; wherein A is a C.sub.2-8 alkylene group; R.sup.1, R.sup.2, and R.sup.3 are independently selected from hydrogen, C.sub.1-4 alkyl, and C.sub.3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, or R.sup.1 is selected from hydrogen, C.sub.1-4 alkyl, and C.sub.3-4 dihydroxyalkyl in which the carbon atom attached to the nitrogen atom does not carry a hydroxyl group and no carbon atom is substituted by two hydroxyl groups, and R.sup.2 and R.sup.3 together with the nitrogen atom to which R.sup.2 and R.sup.3 are attached form a heterocyclic ring, wherein the heterocyclic ring consists of a C.sub.2-6 alkylene group together with the nitrogen atom; and X.sub.1, X.sub.2, and X.sub.3 are independently selected from hydrogen, hydroxyl, NR.sup.1-A-NR.sup.2R.sup.3, halogeno amino, C.sub.1-4 alkoxy, and C.sub.2-8 alkanoyloxy; said composition further comprising a first nucleic acid and a second nucleic acid, wherein the compound of Formula (I) forms a first fluorescent complex including the first nucleic acid, and the compound of Formula (II) forms a second fluorescent complex including the second nucleic acid, and the first fluorescent complex is formed in a non-intact cell and the second fluorescent complex is formed in an intact cell.
2. A composition according to claim 1 in which in Formula (I) at least one of X.sub.1, X.sub.2 and X.sub.3 are NR.sup.1-A-NR.sup.2R.sup.3R.sup.4+(Z.sup.m−).sub.1/m.
3. A composition according to claim 2 in which in Formula (I) X.sub.2, but not X.sub.1 and X.sub.3 is NR.sup.1-A-NR.sup.2R.sup.3R.sup.4+(Z.sup.m−).sub.1/m.
4. A composition according to claim 3 in which in Formula (I) X.sub.1 and X.sub.3 are both hydroxyl.
5. A composition according to claim 1 in which in Formula (I) X.sub.1 and X.sub.3 are both hydrogen.
6. A composition according to claim 1 in which in Formula (I) R.sup.1 is hydrogen.
7. A composition according to claim 1 in which in Formula (I) R.sup.2, R.sup.3, and R.sup.4 are Ci.sub.1-4.
8. A composition according to claim 7 in which in Formula (I) R.sup.2, R.sup.3, and R.sup.4 are methyl.
9. A composition according to claim 1 in which in Formula (I) A is (CH.sub.2).sub.2.
10. A composition according to claim 1 in which the compound of Formula (I) is of Formula (IA). ##STR00010##
11. A composition according to claim 1 in which the compound of Formula (I) is of Formula (IB) ##STR00011##
12. A composition according to claim 1 in which Formula (II) is a 1,5-amino substituted anthraquinone.
13. A composition according to claim 1 in which the ##STR00012##
14. A composition, comprising a mixture of: a compound of Formula (IA) ##STR00013## where (Z.sup.m−) is an anion of charge m, and where m is 1, 2 or 3; together with at least a second fluorochrome or light-emitting compound, wherein the compound is of Formula (IIA) ##STR00014##
15. A composition according to claim 1 in which in Formula (I) X.sub.1, X.sub.2, and X.sub.3 are independently selected from hydroxyl, NR.sup.1-A-NR.sup.2R.sup.3R.sup.4+(Z.sup.m−).sub.1/m, halogeno amino, C.sub.1-4 alkoxy, and C2.sub.2-8 alkanoyloxy.
16. The composition of claim 14, further comprising a first nucleic acid and a second nucleic acid, wherein the compound of Formula (IA) forms a first fluorescent complex including the first nucleic acid, and the compound of Formula (IIA) forms a second fluorescent complex including the second nucleic acid.
17. The composition of claim 14, wherein the compound of Formula (IA) and the compound of Formula (IIA) are each present at a concentration of at most 5 mM.
Description
(1) Embodiments of compounds, fluorescent complexes, methods and detection systems in accordance with the invention will now be described with reference to the accompanying drawings, in which:—
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(16) The invention provides a means of labelling live cells and dead cells using a combination of a permeant dye and an impermeant dye. This combination of dyes can be substantially mutually exclusive. In the sense that, detection of the permeant dye can be associated with the presence of live cells, and the detection of the cell impermeant dye can be associated with the presence of dead cells. This principle is shown in Table 1 below:
(17) TABLE-US-00002 TABLE 1 Positive labelling of live/dead cells through detection of cell permeant/cell impermeant dyes. Cell state Dye B.sup.permeant Dye A.sup.non-permeant live + − dead − +
(18) Thus, the invention provides the capability to associate a live cell with fluorescence from the cell permeant dye, and to associate a dead cell with fluorescence from the cell impermeant dye, since there is little or no “cross channel” interference between the dyes. It will be appreciated that this provides the opportunity to perform numerous advantageous two-colour, two-fluorochrome experiments. Moreover, the present inventors have realised that dye combinations of this type also provide a platform for performing a range of advantageous experiments using one or more further fluorochromes. Table 2 shows without limitation examples of detection systems of this type with reference to the specific cell permeant/cell impermeant dye combination of compound of Formula (IIA)/compound of Formula (IA).
(19) TABLE-US-00003 TABLE 2 Examples of fluorescent probes that can be used with a cell permeant/cell impermeant dye combination and predicted positive or negative staining patterns. Inclusion Inclusion of fluors of fluors with different Cell with overlapping spectral State spectral properties properties deter- Orange Other mined Combination Red probe for fluors by a staining Probe an analyte spec- com- patterns for an in non- trally pound Com- analyte intact cells distinct (IA) pound Com- in (eg an Green for the com- of pound intact Alexa 568 probe anal- bined for- (IA) cells dye- for ysis, with a mula Far (eg Qdot tagged inact for com- (IIA) red 705 nm antibody cells exam- pound (at (at emitting for a (eg ple of 530 >695 nano- disrupted An- of cell formula nm) nm) particle cell nexin surface (IIA) stain- stain- labelled membrane V- ana- analysis ing ing cell) feature) FITC) lytes Live + − + − + or − + or − cell stain Dead − + − + + or − + or − cell stain l
(20) These possible detection systems and others will now fee described in more detail with reference to
(21) One useful consequence of this system is that it is possible to provide a third defection channel using two colour defection in the spectral regions A and B,
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EXAMPLE 1. SYNTHESIS OF COMPOUND OF FORMULA (IIA) [1,5-BIS{[2-(DIMETHYLAMINO)ETHYL]AMINO}ANTHRACENE-9,10-DIONE] AND A COMPOUND OF FORMULA (IB) [1,5-BIS{N-[2-(TRIMETHYLAMINO)ETHYL]AMINO}ANTHRACENE-9,10-DIONE IODIDE]
(26) [1,5-Bis{N-[2-(dimethylamino)ethyl]amino}anthracene-9,10-dione was synthesised according to Example 1 of WO39/85992. Chloroform (2 mL) was added to 1,5-Bis{(2-(dimethylamino)ethyl]amino}anthracene-9,10-dione (58 mg, 0.152 mmol). To the dark purple solution was added MeCN (1 mL) followed by methyl iodide (95 μl, 1.524 mmol). After stirring for 10 mins, a precipitate forms. After stirring for 4 hrs, the volatiles were evaporated. The residue was triturated from chloroform (5 ml), collected by filtration was washed with dichloromethane (20 ml) and diethylether (10 ml). A dark pink solid was isolated (0.09 g, 0.135 mmol, 89% yield). .sup.1H NMR (400 MHZ, d.sub.6-DMSO) δ: 9.69 (2H, t), 7.72 (2H, dd), 7.52 (2H, dd), 7.30 (2H, dd), 3.91 (4H, q), 3.62 (4H, t), 3.18 (18H, s)
EXAMPLE 2. SYNTHESIS A COMPOUND OF FORMULA (IA) [1,5-BIS{[2-(TRIMETHYLAMINO)ETHYL]AMINO}-5,8-DIHYDROXYANTHRACENE-9,10-DIONE IODIDE]
(27) 1,5-Bis{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione was synthesized according to Example 1 of WO99/65992. Chloroform (2 mL) was added to 1,5-Bis{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (44 mg, 0.107 mmol). To the dark purple solution was added MeCN (2 ml) followed by methyl iodide (66.6 μl, 1.067 mmol). After stirring for 1 min a precipitate forms. After stirring for 4 hrs, the volatiles were evaporated. The residue was triturated from chloroform (5 ml), collected by filtration was washed with dichloromethane (20 ml) and diethylether (10 ml). A dark blue solid was isolated. Yield=173-012 (0.05 g, 0.068 mmol, 63.9% yield) .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ: 13.97 (2H, s), 9.77 (2H, br t), 7.49 (2H, d), 7.40 (2H, d), 3.98 (4H, br q), 4.09 (4H, t), 3.19 (18H, s).
EXAMPLE 3 SPECTRAL PROPERTIES OF COMPOUND (IA)
(28) Compound (IA) was synthesized using the principles described in Example 2 and stored at +4° C. as a stock solution of 5 mM compound (IA) dilution in buffer. Absorbance spectra were obtained using a spectrometer and a 20 μM solution of agent dissolved in PSS and measured in a 1 cm path quartz silica cuvette. Fluorescence spectra for a solution of 20 μM compound (IA) in a 1 cm path length semi-micro quartz cuvette were determined by excitation at 633, 589, 534, 488 nm. Fluorescence measurements were made on a Parkin Elmer LS50 spectrofluorometer with slit widths set at 10 nm. The spectrofluoremeter was equipped with a red-sensitive photomultiplier tube (Type R928; Hamamatsu Photonics KK, Japan. Data were accumulated and exported into a spread sheet to correct for the buffer control and to determine emission maxima. The results are shown in
EXAMPLE 4 APPLICATION OF CELL IMPERMEANT PROPERTIES OF COMPOUND (IA) IN DISTINGUISHING BETWEEN LIVE AND DEAD CELLS IN AN ANNEXINV ASSAY FOR THE INDUCTION OF CELL DEATH
(29) This example relates to later stage cell death associated with the translocation of phosphatidylserine molecules from the inner (cytoplasmic) leaflet of the plasma membrane in human B cell lymphoma (DoHH2) cells that have been exposed to a cytotoxic drug (VP-18). Detection of dose-dependent induction of apoptosis was performed using flow cytometry.
(30) We have sought to demonstrate the application of compound (IA) as a cell viability marker determining the non-viable fraction due to cell enhanced membrane permeability as a result of induced apoptosis with an etoposide (VP-16) including the spectral advantages of using a deep-red fluorescent probe from that of other commonly used fluorochromes by using selective excitation.
(31) Compound IA was able to detect with similar accuracy as propidium iodide the non-viable fraction. This fraction showed a dose dependent increase upon increased doses of etoposide. Timed uptakes were done to optimise compound (IA) labelling. Dose modification was also performed to determine optimal concentration of compound (IA).
(32) Reagents
(33) VP-18 (VP-18-213; VEPESID; Etoposide) was provided as a 34 mM stock solution (Bristol Meyers Pharmaceuticals, Syracuse, N.Y.) and stored at 4° C. Fluorescein-conjugated annexin V (annexin V-FITC) was purchased from Pharmingen (Becton Dickinson UK, Oxford, UK.), Propidium iodide (PI) was obtained as a 1 mg/ml solution in H.sub.2O (Molecular Probes Europe, Leiden, The Netherlands). Compound (IA) was formulated in water as a 5 mM solution and stored at 4° C.
(34) Cell Culture and Drug Treatment:
(35) The human follicular B-lymphoma cell lines were used in this study. DoHH2 was a kind gift from Dr J C Kluin-Nelemans [Leiden, The Netherlands], DoHH2 was routinely maintained in RPMI 1640 supplemented with 5% FCS and 100 U ml-1 penicillin, 100 μg ml-1 streptomycin, and 2 mM glutamine. The cells were passaged twice weekly at an initiating density of 5×104 cells ml-1 cultured at 37° C. in a humidified atmosphere of 5% CO2/95% air. Cells were exposed to a range of VP-16 doses (0-2.5 μM) to induce apoptosis [Paul J. Smith, Marie Wiltshire, Sharon Davies, Suet-Feung Chin, Anthony K. Campbell, and Rachel J. Errington (2002). DNA damage-induced [Zn2+]i transients: correlation with cell cycle arrest and apoptosis in lymphoma cells. Am J Physiol Cell Physiol 283 (2): 609-622], Human Jurkat cells were cultured in a similar manner.
(36) Sample Preparation for Annexin-V Labelling;
(37) Samples were prepared for the detection of Annexin V-FITC surface binding to cells undergoing apoptotic changes and co-stained with PI or the compound (IA) to detect loss of plasma, membrane integrity. Samples were prepared according to Vermes et al. Briefly, cell samples (4×10.sup.5 cells/ml) were washed with cold PBS and resuspended in 1× binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl.sub.2) at a concentration of 2×10.sup.5 cells/ml. 100 μl of this solution was transferred to a polystyrene round bottomed flow tube (Falcon) per sample to which 5 μl of Annexin V-FITC and 10 μl PI (50 μg/ml stock) was added as required. Control samples were sham-treated as necessary. Samples were gently vortexed, then incubated in the dark for 15 min at room temperature. 400 μl of 1× binding buffer was added to each tube and samples held on ice for a maximum of 1 hour prior to analysis by flow cytometry.
(38) Flow Cytometry:
(39) A FACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) equipped with a Coherent Enterprise II argon ion laser having 438 nm and multiline UV (351-355 nm) outputs (Coherent, Inc., Santa Clara, Calif.) was used. The Enterprise II laser power was regulated at 30 mW (monitored on the multiline UV output) CELLQuest software (Becton Dickinson Immunocytometry Systems) was used for signal acquisition and analysis. Forward scatter (FSC) and side scatter (SSC) were acquired in linear mode. FITC and PI fluorescent signals derived from 488 nm excitation were detected in logarithmic mode at photomultipliers detecting emissions spectrally selected by optical filters; compound (IA) signal derived from 488 nm excitation was detected in logarithmic or linear mode but could also be detected following excitation at 633 nm using a third laser conveniently incorporated into the optical system. Compound of Formula (IIA) signal derived from excitation at 488 nm was detected in both logarithmic or linear mode. Signals for forward and side scatter and fluorescence were collected for 10,000 cells using the forward light scatter parameter as the master signal. Pulse analysis of fluorescence signals and fluorescence compensation settings were modified to improve recognition of cell subsets in multi-fluor combinations as readily understood in the art. Data are expressed as mean fluorescence intensity (FI) values and are shown in
EXAMPLE 5 PROMINENT NUCLEAR STAINING OF FIXED U-2 OS HUMAN OSTEOSARCOMA CELLS BY A COMPOUND OF FORMULA (IA) DETECTED BY FLUORESCENCE MICROSCOPY
(40) We have sought the property of compound (IA) to effectively target nuclear DNA.
(41) Cell culture: Human osteosarcoma cells U-2 OS (ATCC HTB-96) cells (adherent) were cultured in McCoy's 5a medium supplemented with 10% foetal calf serum (PCS), 1 mM glutamine, and antibiotics and incubated at 37° C. in an atmosphere of 6% CO.sub.2 in air. For fluorescence imaging experiments, cells were grown at a density of 1×10.sup.5 cells ml.sup.−1 as a monolayer in coverglass bottomed chambers (Nunc, 2 Well Lab-Tek II, Fisher Scientific).
(42) Imaging: Following a 24 hour period cells were then fixed with 4% paraformaldehyde in PBS for 15-30 min at room temperature. No washing step is required. Compound (IA) was used in a manner appropriate to being the final staining procedure, after any treatment. Compound (IA) was added directly at 20 μM in to a 0.5 ml PBS overlay of the adherent cells. Cells were directly viewed using wide-field fluorescence microscopy. Chambers were placed onto an Axiovert 100 microscope (Carl Zeiss, Welwyn Garden City, UK and using a 40×, 1.3 NA oil immersion plan apochromat lens) fluorescence images (Ex: 620/60 nm; Em 700/75 nm) nm captured using an ORCA-ER CCD camera (Hamamatsu, Reading, UK) and MetaMorph (MDS, USA) acquisition software. Cells showed the localization of high levels of compound (IA) in the nucleus. The compound (IA) staining of nuclei were segmented using a simple threshold algorithm which depict the nuclei, and provide binary or mask information of each nucleus (object) localization. The original compound (IA) nuclear localisation and after segmentation are shown. Results are shown in
EXAMPLE 6. THE STAINING OF CELLS WITH COMPROMISED MEMBRANES BY A COMPOUND OF FORMULA (IA) ALLOWS FOR THE IDENTIFICATION BY NEGATIVE STAINING OF INTACT CELLS
(43) This example shows the analysis of cell death induction by staurosporine in populations of human Jurkat cells analysed by flow cytometry. We have sought to demonstrate the application of compound (IA) as a cell viability marker determining the non-viable fraction due to cell enhanced membrane permeability as a result of induced cell death with staurosporine.
(44) Cell Culture and Drug Treatment:
(45) A Jurkat cell line was used in this study Jurkat cultures was routinely maintained in RPMI 1840 supplemented with 10% PCS and 100 U ml-1 penicillin, 100 μg ml-1 streptomycin, and 2 mM glutamine. The cells were passaged twice weekly at an initiating density of 5×10.sup.4 cells ml.sup.−1 cultured at 37° C. in a humidified atmosphere of 5% CO2/95% air. Cells were set at 0.5×10.sup.5 cells/ml, 5 ml per flask. Cells were exposed to 0 and 2 μM staurosporine for 24 hours under standard culture conditions to induce cell death. Compound (IA) (3 μM) from a 5 mM stock was added to each sample and analysed by flow cytometry.
(46) Flow Cytometry:
(47) A FACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) equipped with a Coherent Enterprise II argon ion laser having 488 nm and multiline UV (361-355 nm) outputs (Coherent, Inc., Santa Clara, Calif.) was used. The Enterprise II laser power was regulated at 30 mW (monitored on the multiline UV output) CELLQuest software (Becton Dickinson Immunocytometry Systems) was used for signal acquisition and analysis. Forward scatter (FSC) and side scatter (SSC) were acquired in linear mode. Compound (IA) fluorescent signals derived from 488 nm excitation were detected in logarithmic mode at FL3 695LP Signals for forward and side scatter and fluorescence were collected for 10,000 cells using the forward light scatter parameter as the master signal. Data are displayed as contour plots fluorescence intensity (695LP) for compound (IA) against forward scatter signal to indicate cell size. Results are shown in
EXAMPLE 7. THE EFFECT OF INCUBATION OF HUMAN B CELL LYMPHOMA (SU-DHL-4) CELLS WITH A COMPOUND OF FORMULA (IA) REVEALING THE LOW TOXICITY OF THE CELL IMPERMEANT DYE INDICATING AN ADVANTAGEOUS PROPERTY FOR INCORPORATION INTO LONG TERM FIVE CELL INCUBATION STUDIES FOR THE DETERMINATION OF THE ACCRUAL OF CELL DEATH ASSOCIATED WITH A GIVEN TREATMENT
(48) Cell Culture and Treatment:
(49) A human follicular 8-lymphoma cell lines were used in this study. SU-DHL-4 was routinely maintained in RPMI 1640 supplemented with 10% FCS and 100 U ml-1 penicillin, 100 μgml.sup.−1 streptomycin, and 2 mM glutamine. The cells were passaged twice weekly at an initiating density of 5×10.sup.4 cells ml.sup.−1 cultured at 37° C. in a humidified atmosphere of 6% CO.sub.2/95% air. Cells were set at 0.5×10.sup.5 cells/ml at 5 ml per flask. Each culture was continuously exposed to one of three compound (IA) closes (0, 3 and 10 μM) for 96 hours under standard culture conditions. At time (t) 0, 24, 48, 72 and 96 hours cell density was determined by Coulter counting of 0.4 ml samples from each flask. Data are displayed as increase in relative cell number (Nt/N0) (695LP) against time (hours) for the given concentration of compound (IA). The results are shown in
EXAMPLE 8. EARLY STAGE IN HUMAN JURKAT CELL DEATH ASSOCIATED WITH THE LOSS OF MITOCHONDRIAL MEMBRANE POTENTIAL IN RESPONSE TO THE APOPTOSIS INDUCING AGENT STAUROSPORINE
(50) The incorporation of a preferred cell impermeant dye (compound of Formula (IA)) into typical multi-parameter analyses using other fluorescent reagents with properties of interest in reporting the loss of cellular integrity with advantages for the co-incorporation into assays to provide a means of distinguishing intact and damaged cells.
(51) In this example, the dye JC-1 (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazol-carbocyanine iodide) is a lipophilic fluorescent cation that incorporates into the mitochondrial membrane, where it can form aggregates due to the physiological maintenance membrane potential of mitochondria. Aggregation modifies the fluorescence properties of JC-1 leading to a shift from green to orange fluorescence. Flow cytometry or imaging was used to monitor the decrease of the orange fluorescence and an increase of the green fluorescence allowing apoptotic cells to be distinguished from non-apoptotic cells. Here the further incorporation of the preferred cell impermeant dye with red but not orange fluorescence properties allows for the co-identification of those cells already in late stages of cell death associated with loss of membrane integrity providing a finer resolution of the stages of cell death not previously attainable.
(52) Cell Culture and Drug Treatment:
(53) A Jurkat cell line was used in this study Jurkat cultures was routinely maintained in RPMI 1640 supplemented with 10% PCS and 100 U ml-1 penicillin, 100 μg ml-1 streptomycin, and 2 mM glutamine. The cells were passaged twice weekly at an initiating density of 5×10.sup.4 cells ml.sup.−1 cultured at 37° C. in a humidified atmosphere of 5% CO2/95% air. For the assay, cells were set at 0.5×10.sup.5 cells/ml, 1 ml per well. Cells were exposed to 0 (control conditions) and 1 μM staurosporine for 4 hours. Cells were washed and exposed to JC-1 (RPM). Compound (IA) (3 μM) from a 5 mM stock was added to each sample. These were then analysed by flow cytometry or placed into a Nunc, 2 Well Lab-Tek II, (Fisher Scientific) and analysed by three channel confocal microscopy.
(54) Multi-Parameter how Cytometry of Staurosporine Treated Cells
(55) A FACS Vantage flow cytometer (as for example above). Forward scatter (FSC) and side scatter (SSC) were acquired in linear mode. JC-1 two parameter fluorescent signal were derived from 488 nm excitation were detected in logarithmic mode Green J-monomer was detected using FL1 (530/30 nm emission); Orange J-aggregate was detected using FL2 (585/42 nm emission). Compound (IA) fluorescent signals also derived from 488 nm excitation were detected in logarithmic mode at FL3 at 695LP. Signals for forward and side scatter and fluorescence were collected for 10,000 cells using the forward light scatter parameter as the master signal. Results shown in
(56) Multi-parameter imaging of staurosporine treated cells. The scanning unit was a BioRad Radiance MP system (BioRad Microscience, Hemel Hempstead, UK) linked to a Nikon Eclipse TE300 inverted microscope, using a planapo 60×/1.4 NA oil immersion lens. Three channel, three-dimensional (3D) (x,y,z) images were collected using a confocal configuration (pinhole closed). All channels green (J-monomer excitation at 488 nm emission at 500-530 nm); orange (J-aggregate excitation at 488 nm emission at 590/70; and red (compound (IA)) (non-viable cell marker excitation at 637 nm emission at 660LP) were collected simultaneously. The 3D image sequence were processed into single maximum projection images, and all three channels displayed as J-monomer, J-aggregate and DNA nucleus cells. Results are shown in
(57) We have sought the exploitation of multi-fluorochome applications exploiting the concept of signal extinction by target competition between the preferred cell impermeant dye and a second preferred cell permeant dye. The spectral separation provides for exclusive signals arising from only one dye within any given cell and therefore allows for their simple integration into existing multi-fluorochome assays with higher levels of polychromatic analyses readily understood within the field.
EXAMPLE 9. COMBINATION TRACKING OF CALL STATUS. THE LABELING OF CELLULAR POPULATIONS WITH THE COMPOUND OF FORMULA (IA) PLUS COMPOUND (IIA) TO DEMONSTRATE NO CO-LABELLING OF SUB-POPULATIONS IN DOHH2 CULTURES TREATED WITH VP-16 TO DERIVE AN ASSAY ACCOUNTING FOR VIABLE/ARRESTED (COMPOUND (IIA) POSITIVE) AND DAMAGED (COMPOUND (IA) POSITIVE) CELLS
(58) Cell Culture and Drug Treatment:
(59) A human follicular B-lymphoma cell line was used in this study. DoHH2 was routinely maintained in RPMI 1840 supplemented with 5% FCS and 100 U ml-1 penicillin, 100 μgml.sup.−1 streptomycin, and 2 mM glutamine. The cells were passaged twice weekly at an initiating density of 5×10.sup.4 cells ml.sup.−1 cultured at 37° C. in a humidified atmosphere of 5% CO.sub.2/95% air. Cells were set at 5×10.sup.5 cells/per ml and exposed to VP-16 doses (0.25 μM) to induce apoptosis, 20 μM Compound (IIA) and 4 μM compound (IA) were added to 1 ml of cells and incubated under standard culture conditions for 10 minutes. The samples were analysed by flow cytometry.
(60) A FACS Vantage flow cytometer (as for example above) was used. Compound (IIA) and compound (IA) fluorescent signals derived from 488 nm excitation were defected in linear mode at FL2 for Compound (IIA) (585/42 nm filters); compound (IA) signal derived from 488 nm were detected in linear mode at FL3 with a 695LP filter and a FL 1/2 580 nm SP dichroic SP to determine scatter properties. Signals for forward and side scatter and fluorescence were collected for 10,000 cells using the forward light scatter parameter as the master signal. Data are expressed as contour plots and are shown in