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C3N4 Nanosheet Modified Microwell Array with Enhanced Electrochemiluminescence for Total Analysis of Cholesterol at Single Cells Jingjing Xu, Depeng Jiang, Yanling Qin, Juan Xia, Dechen Jiang, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04635 • Publication Date (Web): 02 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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Analytical Chemistry

C3N4 Nanosheet Modified Microwell Array with Enhanced Electrochemiluminescence for Total Analysis of Cholesterol at Single Cells

Jingjing Xu1, Depeng Jiang2, Yanling Qin2, Juan Xia2, Dechen Jiang1*, Hong-Yuan Chen1

1 State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China 2 Department of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China

Corresponding Author Phone/Fax: 086-25-83594846 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract:

Here,

g-C3N4

nanosheet

modified

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microwell

array

providing

enhanced

electrochemiluminescence (ECL) and better visible sensitivity was prepared to simultaneously analyze total (membrane and intracellular) cholesterol at single cells. The detection limit for ECL visualization of hydrogen peroxide at microwell array was improved to be 500 nM that guaranteed the detection of low concentrated cholesterol at single cells in parallel. To achieve single cell cholesterol analysis, the individual cells cultured at microwell array was exposed to cholesterol oxidase generating hydrogen peroxide for luminescence analysis of membrane cholesterol; and then, treated with triton X-100, cholesterol esterase and cholesterol oxidase to produce hydrogen peroxide from intracellular cholesterol for luminescence determination. The observation of the luminescence spots at microwells in these two steps confirmed the co-detection of membrane and intracellular cholesterol at single cells.

The inhibition of intracellular

acyl-coA/cholesterol acyltransferase (ACAT) resulted in less intracellular cholesterol storage (less luminescence) and more membrane cholesterol (more luminescence). The correlation of the luminescence intensity with the amount of cholesterol confirmed that our assay could simultaneously monitor membrane and intracellular cholesterol pools at different cellular states, which should offer more information for the study of cholesterol-related pathways at single cells.


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Introduction. Free cholesterol is a largely hydrophobic molecule with polar head group that is mainly found in plasma membrane. The ester of cholesterol, named as cholesteryl ester, is much less polar than free cholesterol, and packed into intracellular lipid particles.1-3

Classically, total

cholesterol at the cells means the total amount of free cholesterol at plasma membrane and intracellular cholesterol (mainly cholesteryl ester).4-7

The reciprocal conversion between

membrane cholesterol and intracellular cholesterol facilitates the cholesterol storage inside the cells and cholesterol trafficking to cellular membrane.8-12

The disorders in the conversion have

been proved to be associated with various disease, such as atherosclerosis and Niemann–Pick disease.13-15 Therefore, the co-determination of membrane and intracellular cholesterol at the cells, especially one cell, to obtain the information about total cholesterol is significant for the full understanding of cholesterol homeostasis. Previously, our group utilized luminol electrochemiluminescence (ECL) for the determination of plasma membrane cholesterol at single cells, which included the activation of membrane cholesterol, the reaction of active cholesterol with aqueous cholesterol oxidase producing hydrogen peroxide and the generation of ECL from aqueous luminol and hydrogen peroxide.16,17

The luminescence intensity was proved to be correlated with the amount of

membrane cholesterol so that it could be quantitatively measured. To increase the analysis throughput, ECL from the cells was recorded using charge-coupled device (CCD) to realize parallel determination of membrane cholesterol at single cells.18 In this analysis, cholesterol oxidase and luminol parked at the small space between the bottom surface of the cells and the electrode, which reacted with membrane cholesterol and generated ECL. This parallel analysis of multiple cells increased the analysis rate, however, the volume of the space under the cells was not controlled, and thus, the quantification of membrane cholesterol using luminescence intensity was not accurate. Recently, we cultured individual cells at Au nanoparticles (Au-NPs) deposited indium tin oxides (ITO) regions in the microwells and realized the quantitative analysis of intracellular glucose at single cells using ECL imaging.19

During the analysis, the detergent, triton X-100,

was introduced at the cells to break cellular membrane. Glucose in the cytosol was released and reacted with aqueous glucose oxidase in the microwells to generate hydrogen peroxide and the following luminol ECL. Since the volume of the microwells was fixed, the amount of glucose in



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single cells was analyzed quantitatively. The difficulty to expand this approach on the parallel detection of cellular cholesterol was ascribed to the relatively poor visible sensitivity (5 µM for hydrogen peroxide), which was not sensitive enough to detect low concentrated cholesterol at the cells. Graphitic carbon nitride (g-C3N4) nanosheets, similar to graphene and graphene oxide, had high affinity to aromatic compounds, such as luminol, through π-π stacking.20 observed

the

overlapping

of

the

absorption

spectrum

of

Researchers have

g-C3N4 nanosheets

chemiluminescence spectrum of luminol-hydrogen peroxide at 425 nm.

with

Therefore, g-C3N4

nanosheets could partially adsorb ECL emission from luminol-hydrogen peroxide, which catalyzed hydrogen peroxide to generate more •OH radical for enhanced ECL.21

In this note,

g-C3N4 nanosheets were attempted to be modified at Au-NPs deposited ITO regions in the microwells to decrease the visible limit for the quantitative analysis of cholesterol at the cells. Meanwhile, immediately after the measurement of membrane cholesterol, the strategy for the analysis of glucose in cytosol will be adapted so that intracellular cholesterol at single cells could be determined at the same cell. The collection of the quantitative information about membrane and intracellular cholesterol at one cell should provide more data for the study of cholesterol trafficking. The detection procedure for the co-measurement of membrane and intracellular cholesterol at one cell was clarified in Figure 1. Individual cells were retained at g-C3N4 nanosheets modified Au NPs/ITO regions in the cell-sized microwells.

In the first step, cholesterol oxidase was

introduced and reacted with cholesterol at plasma membrane to generate hydrogen peroxide inducing ECL for the quantitative determination of membrane cholesterol. In the second step, triton X-100 was employed to break the cell membrane.

Simultaneously, the addition of

cholesterol oxidase and cholesterol esterase reacted with intracellular cholesterol (mainly cholesteryl esters) in the cytosol to produce hydrogen peroxide and the following ECL. As the result, two luminescence intensities associated with membrane and intracellular cholesterol at one cell were collected serially using ECL imaging to investigate their correlation.

Experimental Section.


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Chemical and cell culture. The SU-8 photoresist and the developer were purchased from Microchem Corp (Newton, MA).

The compound 8-amino-5-chloro-7-phenylpyrido [3, 4-d]

pyridazine-1, 4(2H, 3H)-dione (L012) was obtained from Wako Chemical, Inc. (Richmond, VA). All other chemicals were from Sigma-Aldrich, unless indicated otherwise. Hela cells were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science of Chinese Academy of Science (Shanghai, China). Hela cells were seeded in DEME/high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% antibotics (penicillin/streptomycin).

Cultures were maintained at 37°C under a humidified

atmosphere containing 5% CO2. Modification of g-C3N4 nanosheets on Au-NPs deposited ITO surface with microwells. The cell-sized microwells with the diameter of 30 µm and the depth of 30 µm were fabricated on ITO slides. Au nanoparticles (Au-NPs) were deposited on the ITO slide with cell-sized microwells following our previous approach.19

The g-C3N4 nanosheets were prepared as reported.22

g-C3N4 nanosheets were dispersed in ethanol and sonicated sufficiently.

The

Then, 100 µL of 0.2

µg/mL g-C3N4 nanosheets suspension was dropped on the Au-NPs deposited ITO slide and kept for 5 min in the freezer. This process was repeated for three times to introduce sufficient g-C3N4 nanosheets on ITO slide. Luminescence imaging. The luminescence imaging was conducted following our previous work.19 Briefly, the dispersed individual cells in 10 mM phosphate buffer saline (PBS, pH 7.4) with 200 µM L012 was added into the cell chamber and loaded into the cell-sized microwells through self-gravity.

The loading efficiency was over 80%. The extra cells outside the wells were

gently washed away using fresh PBS before their adherence on ITO slide. The microwells without the cell or with more than one cell were excluded in the following luminescence analysis. The ITO slide with 64 cells/microwells was employed as the working electrodes.

An

Ag/AgCl electrode and a Pt wire were connected as the reference and counter electrodes, respectively. The solution for luminescence imaging was 10 mM PBS (pH 7.4) with 200 µM L012. A switching of the potential between 1 V (2 s) and -1 V (0.5 s) was continuously applied on ITO slide using a voltage generator (DG 1021, Rigol, China) to induce the luminescence at room temperature.

The luminescence was recorded using EM CCD (Evolve, Photometrics,

Tucson, AZ). The images were analyzed using Image J software.



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Results and Discussions. ECL Imaging of aqueous hydrogen peroxide with better detection limit.

The previous

experiments suggested that the concentration of hydrogen peroxide produced from membrane cholesterol at one cell was as low as a few micromolar in the microwells. Accordingly, ECL visualization of hydrogen peroxide with this sensitivity was critical to realize the parallel and quantitative determination of cellular cholesterol.

For this aim, the Au-NPs modified ITO

surface was further coated with g-C3N4 nanosheets to enhance the luminescence intensity from luminol and hydrogen peroxide. L012, a luminol analog with enhanced luminescence, was used to induce ECL with aqueous hydrogen peroxide.

The luminescence intensity from g-C3N4

nanosheets modified Au NPs/ITO electrode was approx. 4 fold of that without g-C3N4 nanosheets, as shown in Figure S1 (supporting information). The enhancement of ECL should permit the visualization of lower concentration of hydrogen peroxide in the microwells. The g-C3N4 nanosheets coated ITO surface with the microwells was applied to visualize aqueous hydrogen peroxide in 10 mM PBS (pH 7.4) with 200 µM L012. The bright-field image of the modified ITO surface with the microwells was shown in Figure S2 A (supporting information).

Following the protocol developed before,19 the luminescence from these

microwells were imaged before and after the introduction of hydrogen peroxide from 500 nM to 50 µM. The luminescence difference was calculated and false-colored, as shown in Figure 2A and S2 B-G (supporting information). More luminescence observed after the addition of 500 nM hydrogen peroxide suggested that C3N4 nanosheets modified ITO electrode could induce the visible luminescence in presence of nanomolar hydrogen peroxide.

As compared with the

visualization limit of 5 µM hydrogen peroxide using Au NPs modified ITO surface, 10-fold improvement in the limit was obtained from the g-C3N4 nanosheets modified ITO slide, which was attributed to the high affinity of g-C3N4 nanosheets to luminol and the excellent catalysis of g-C3N4 nanosheets for luminol- hydrogen peroxide system. The linear relationship between luminescence intensity and the concentration of hydrogen peroxide in Figure 2B suggested that this new platform should offer the quantitative measurement of low concentrated hydrogen peroxide at single cells.

The relative standard deviation of

luminescence observed at these 64 microwells in presence of 50 µM hydrogen peroxide was 6.2%,


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which was less than our previous deviations of 17.6% from Au-NPs deposited ITO slides and 16.5% from bare ITO slides. The more uniform luminescence might be attributed to sheet structure of g-C3N4 that was fully covered at ITO regions in microwells.

Both of the

improvements in the visible sensitivity and luminescence uniformity will benefit the following cholesterol analysis at single cells. Imaging of membrane and intracellular cholesterol. To analyze membrane cholesterol at single cells, the cells were pretreated with low ion strength buffer (0.5 mM PBS with 310 mM sucrose, pH 7.4) at 37°C for 1 hour that activated cholesterol at plasma membrane.6

Figure S3

(supporting information) showed the bright field image of 64 microwells loaded with individual cells. The luminescence images from these cells/microwells were recorded before and after the introduction of 1U/ml cholesterol oxidase, which reacted with active membrane cholesterol to generate hydrogen peroxide for ECL, as listed in Figure S4A and B (supporting information). After extracting the background luminescence, more luminescence was observed at the microwells, as exhibited in Figure 3A, suggesting the visualization of membrane cholesterol at single cells. To exclude the possible contribution of intracellular reactive oxygen species on the luminescence, the control experiment was performed at the cells in absence of glucose oxidase. No obvious change in the luminescence was observed at the microwells before and after the introduction of triton X-100 only, which confirmed that the luminescence increase observed at the microwells was attributed to hydrogen peroxide generated from the reaction of intracellular glucose and glucose oxidase. This successful collection of luminescence from the individual cells confirmed that the sensitive C3N4 nanosheets modified ITO surface could be applied for the parallel measurement of membrane cholesterol. Since the contact area of the cells at ITO surface induced varied area of ITO region exposed to the solution, the luminescence intensity from each microwell might be varied. To minimize this deviation, the luminescence intensities at the cells/microwells were rationed with those from 50 µM aqueous hydrogen peroxide in the microwells so that the effect of exposed ITO regions on luminescnece was excluded. Figure 3B summarized the relative luminescence intensities from 156 individual cells.

The averaged relative luminescence from membrane cholesterol was

calculated to be 0.28 ± 0.10, which suggested ~ 13 µM hydrogen peroxide generated in the microwell. Taking in account of the microwell volume, membrane cholesterol at one cell was



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estimated to be 0.26 femtomoles, which was close to the literature and our previous measurement.6, 17 This result supported that our new ECL imaging system could be applied to quantitatively measure membrane cholesterol. The relative standard deviation was calculated to be 35.7% exhibiting high cellular heterogeneity on membrane cholesterol, which was similar to our previous results also.16, 17 Afterwards, the measurement of intracellular cholesterol at the same cell was attempted. Experimentally, after washing the chamber and refreshing the solution, a cocktail of cholesterol oxidase, cholesteryl esterase and triton X-100 was introduced into the buffer to lyse the cellular membrane and react with intracellular cholesterol. During the reaction, cholesteryl ester was converted in cholesterol by cholesteryl esterase. Free cholesterol in the cytosol and cholesterol generated from intracellular cholesteryl ester were reacted with cholesterol oxidase to produce hydrogen peroxide.

Following our protocol for the analysis of intracellular glucose,19 two

consecutive images were recorded with the exposure time of 60 s immediately after the introduction of a cocktail with cholesterol oxidase, cholesteryl esterase and triton X-100, as shown in Figure S4 C and D (supporting information). The luminescence difference from these two images was shown in Figure 3C. The visualization of luminescence spots at microwells after the reaction with the enzymes supported the visualization of intracellular cholesterol at single cells. Figure 3D exhibited the relative luminescence intensity from intracellular cholesterol at 156 individual cells calibrated by the luminescence intensity from 50 µM hydrogen peroxide. The averaged relative luminescence was calculated to be 0.19 ± 0.06 suggesting ~ 0.17 femtomoles intracellular cholesterol. The relative standard deviation was calculated to be 31.5% revealing high difference in intracellular cholesterol at the first time. The averaged relative luminescence from intracellular cholesterol was smaller than that from membrane cholesterol, which clarified less intracellular cholesterol than membrane cholesterol from the cells.

This result was

consistent with the conclusion collected from the cell population.23 To investigate the correlation between membrane and intracellular cholesterol, both of them were summed to be 0.48 ± 0.13. The relative standard deviation dropped to 25.5% indicating less cellular heterogeneity on total cholesterol. It was unclear whether this decrease in the deviation suggested the relative uniformity on total cholesterol, and thus, a larger pool of the cells and more cell types needed to be investigated for the understanding of cholesterol distribution.


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Nevertheless, to the best of our knowledge, this was the first report to reveal the association between membrane cholesterol and intracellular cholesterol at single cells. Analysis of membrane and intracellular cholesterol at the cells with different cholesterol state. The cells were pretreated with Sandoz 58035 overnight at 37°C under a humidified atmosphere containing 5% CO2 to inhibit intracellular ACAT so that the intracellular esterification of cholesterol was inhibited and more free cholesterol was accumulated at plasma membrane.24 At this cellular state, intracellular cholesterol decreased and membrane cholesterol increased. Following the same imaging procedures, the luminescence images associated with membrane and intracellular cholesterol at ACAT-inhibited cells were shown in Figure 4 A and C. The raw luminescence images were listed in Figure S5 (supporting information). The visualization of luminescence in both of the images confirmed the analysis of membrane and intracellular cholesterol at ACAT inhibited cells. The relative luminescence intensities from all the cells were shown in Figure 4 B and D.

The average luminescence ratio of membrane and intracellular

cholesterol at ACAT inhibited cells was 0.33 ± 0.14 and 0.09 ± 0.05, respectively, which presented 0.3 femtomoles membrane cholesterol and 0.08 femtomoles intracellular cholesterol.

The

relative standard deviations from membrane and intracellular cholesterol were calculated to be 42.4% and 55.6%.

As compared with non-inhibited cells, the relative luminescence from

membrane and intracellular cholesterol increased and decreased, respectively. These changes were consistent the alteration of cholesterol homeostasis after the inhibition of ACAT. The correlation of the luminescence intensity with the amount of cholesterol confirmed that our assay could simultaneously monitor the intracellular and membrane cholesterol pool at different cellular states. The sum of membrane and intracellular cholesterol exhibited that the total cholesterol at ACAT inhibited cells was 0.42 ± 0.16, which was close to that at non-inhibited cells (0.48 ± 0.13). The similarity indicated that the inhibition of ACAT regulated the distribution of cholesterol at membrane and cytosol, but did not alter the total cholesterol significantly.

Meanwhile, the

deviation of the total cholesterol at single cells was calculated to be 38.1%, which was slightly less than that of membrane and intracellular cholesterol. The further investigation at more cells will be performed to obtain the statistical data.



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Conclusion. In conclusion, total cholesterol, including membrane and intracellular cholesterol, at single cells was analyzed in parallel using ECL imaging. The correlation of the luminescence intensity with the amount of cholesterol confirmed that our strategy could simultaneously measure the membrane and intracellular cholesterol at different cellular states.

Large deviations on

intracellular and membrane cholesterol were observed indicating high cellular heterogeneity on cellular cholesterol homeostasis.

More detail work is needed to investigate cholesterol

distribution at sub-cellular compartments to elucidate the intracellular cholesterol trafficking and understand the cellular heterogeneity on cholesterol distribution.

ACKNOWLEDGEMENTS. This work was supported by the 973 Program (2013 CB933800), the National Natural Science Foundation of China (nos. 21327902, 21135003 and 81650003).

Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Luminescence intensity of C3N4 nanosheet modified ITO regions, more luminescence images.


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References 1. Simons, K.; Ikonen, E. Science 2000, 290, 1721-1726. 2. Maxfield, F. R.; Wustner, D. J. Clin. Invest. 2002, 110, 891-898. 3. Liscum, L.; Munn, N. J. BBA. Mol. Cell Biol. L. 1999, 1438, 19-37. 4. Radhakrishnan, A.; McConnell, H. P. Natl. Acad. Sci. USA 2005, 102, 12662-12666. 5. Lange, Y.; Steck, T. L. Prog. Lipid. Res. 2008, 47, 319-332. 6. Lange, Y. J. Lipid. Res. 1991, 32, 329-339. 7. Ali, M. R.; Cheng, K. H.; Huang, J. P. Natl. Acad. Sci. USA 2007, 104, 5372-5377. 8. Steck, T. L.; Lange, Y. Trends. Cell. Biol. 2010, 20, 680-687. 9. Radhakrishnan, A.; McConnell, H. M. Biochemistry 2000, 39, 8119-8124. 10. Lange, Y.; Matthies, H. J. G. J. Biol. Chem. 1984, 259, 4624-4630. 11. Maxfield, F. R.; Mondal, M. Biochem. Soc. Trans. 2006, 34, 335-339. 12. Ikonen, E. Nat. Rev. Mol. Cell Biol. 2008, 9, 125-138. 13. Maxfield, F. R.; Tabas, I. Nature 2005, 438, 612-621. 14. Ikonen, E. Physiol. Rev. 2006, 86, 1237-1261. 15. Berger, S.; Raman, G.; Vishwanathan, R.; Jacques, P. F.; Johnson, E. J. Am. J. Clin. Nutr. 2015, 102, 276-294. 16. Tian, C.; Zhou, J.; Wu, Z.-Q.; Fang, D.; Jiang, D. Anal. Chem. 2014, 86, 678-684. 17. Ma, G.; Zhou, J.; Tian, C.; Jiang, D.; Fang, D.; Chen, H.Y. Anal. Chem. 2013, 85, 3912-3917. 18. Zhou, J.; Ma, G.; Chen, Y.; Fang, D.; Jiang, D.; Chen, H.Y. Anal. Chem. 2015, 87, 8138-8143. 19. Xu, J.; Huang, P.; Qin, Y.; Jiang, D.; Chen, H.Y. Anal. Chem 2016, 88, 4609-4612. 20. Dong, F.; Wu, L.; Sun, Y.; Fu, M.; Wu, Z.; Lee, S. C. J. Mater. Chem. 2011, 21, 15171-15174. 21. Yu, H.; He, Y.; Li, W.; Duan, T. Sensor. Actuat. B-Chem. 2015, 220, 516-521. 22. Zhang, X.; Xie, X.; Wang, H.; Zhang, J.; Pan, B.; Xie, Y. J. Amer. Chem. Soc. 2013, 135, 18-21. 23. Lange, Y. J. Lipid Res. 1992, 33, 315–321.

24. Warner, G. J.; Stoudt, G.; Bamberger, M.; Johnson, W. J.; Rothblat, G. H. J. Biol. Chem. 1995, 270, 5772-5778.



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Figures and Captions. Figure 1: The process for the analysis of membrane and intracellular cholesterol at single cells. Figure 2: (A) The typical luminescence image of 500 nM hydrogen peroxide at g-C3N4 nanosheets modified Au NPs/ITO regions in cell-sized microwells. The luminescence intensity was the luminescence ratio before and after the addition of hydrogen peroxide. The solution was 10 mM PBS with 200 µM L012. (B) the correlation of luminescence intensity with the concentrations of hydrogen peroxide. The error bar presented the standard deviation measured from 64 microwells on the ITO slide. Figure 3: (A) The typical luminescence image of membrane cholesterol at single Hela cells in microwells. The luminescence intensity was the luminescence difference before and after the addition of cholesterol oxidase; (B) the relative luminescence intensities from 156 cells for the analysis of membrane cholesterol; (C) the typical luminescence image of intracellular cholesterol at single Hela cells in microwells; (D) the relative luminescence intensities from 156 cells for the analysis of intracellular cholesterol. The luminescence from 50 µM aqueous hydrogen peroxide was used as the calibrator. The solution was 10 mM PBS with 200 µM L012. The horizontal line in Figure (B) and (D) represented the number of the individual cells. The ECL intensity was the mean luminescence value from each microwell. Figure 4: (A) The typical luminescence image of membrane cholesterol at single ACAT inhibited Hela cells in microwells region. The luminescence intensity was the luminescence difference before and after the addition of cholesterol oxidase; (B) the relative luminescence intensities from 156 cells for the analysis of membrane cholesterol; (C) the typical luminescence image of intracellular cholesterol at single ACAT inhibited Hela cells in microwells; (D) the relative luminescence intensities from 156 cells for the analysis of intracellular cholesterol. The solution was 10 mM PBS with 200 µM L012. The horizontal line in Figure (B) and (D) represented the number of the individual cells. The ECL intensity was the mean luminescence value from each microwell.


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Figure 1.

Triton X-100

Cholesterol Oxidase

Cholesterol esterase Cholesterol oxidase

Membrane cholesterol Intracellular cholesterol

EM CCD


EM CCD


Figure 2.

A

B Luminescence /a.u.

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400

200

0 0

25 Conc. /µM


50

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Figure 3.

A Luminescence ratio

0.8

B

0.6 0.4 0.2 0.0 0

50

100

150

Cell

0.8

C Luminescence ratio

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D

0.6 0.4 0.2 0.0 0

30

60

90 Cell


120

150


Figure 4.

A

B

Luminescence ratio

0.8 0.6 0.4 0.2 0.0 0

50

100

150

100

150

Cell

0.8

C Luminescence ratio

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D

0.6 0.4 0.2 0.0 0

50 Cell


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C3N4 Nanosheet Modified Microwell Array with ... - ACS Publications

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