Localized Gene Expression Analysis during Sprouting Angiogenesis

Subscriber access provided by CMU Libraries - http://library.cmich.edu

Letter

Localized gene expression analysis during sprouting angiogenesis in mouse embryoid bodies using a double barrel carbon probe Hidenori Ito, Yuji Nashimoto, Yuanshu Zhou, Yasufumi Takahashi, Kosuke Ino, Hitoshi Shiku, and Tomokazu Matsue Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04338 • Publication Date (Web): 27 Nov 2015 Downloaded from http://pubs.acs.org on December 7, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Localized gene expression analysis during sprouting angiogenesis in mouse embryoid bodies using a double barrel carbon probe Hidenori Ito, † Yuji Nashimoto, †,‖ Yuanshu Zhou, ‡ Yasufumi Takahashi, †,‡,§,‖ Kosuke Ino, † Hitoshi Shiku, †,* Tomokazu Matsue †,‡,** †Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan ‡WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan §PRESTO, JST, Saitama 332-0012, Japan ABSTRACT: The mouse embryonic stem (ES) cell-derived angiogenesis model is widely used as a 3D model, reproducing cellcell interactions in the living body. Previously, many methods to analyze localized cellular function, including in situ hybridization and laser capture microdissection, have been reported. In this study, we achieved a collection of localized cells from the angiogenesis model in hydrogel. The gene expression profiles of the endothelial cells derived from mouse ES cells were evaluated. First, we collected localized cells from the live tissue model embedded in hydrogel using the double barrel carbon probe (DBCP) and quantified mRNA expression. Second, we found that vascular marker genes were expressed at a much higher level in sprouting vessels than in the central core of the embryoid body because the cells in sprouting vessels might significantly differentiate into endothelial linages, including tip/stalk cells. Third, the gene expression levels tended to be different between the top and middle regions in the sprouting vessel due to the difference in the degree of differentiation in these regions. At the top region of the vessel, both the tip and stalk cells were present. The cells in the middle region became more mature. Collectively, these results show that DBCP is very useful for analyzing localized gene expression in cells collected from 3D live tissues embedded in hydrogel. This technique can be applied to comprehensive gene expression analyses in the medical field.

Angiogenesis, the formation of new vessels from the existing stable vessel structure, is essential for the development of normal tissues and as well as of cells with special characteristics in various diseases including cancer.1,2 Vascular endothelial growth factors (VEGF-A) mainly control the formation and function concerning angiogenesis and regulate the coordinated behavior of endothelial cells, including the processes of migration and proliferation.3-5 The elucidation of the mechanism underlying angiogenesis is expected to be a breakthrough for the medical field in the future. Proangiogenic growth factors such as VEGF-A and other cytokines control angiogenesis by guiding filopodial extensions from specialized endothelial cells that are situated at the tips of the sprouting vessels.3-5 The tip cell has dynamic filopodia and is considered the route of sprouting vessels with Dll4-Notch signals.3,4,6-11 The signal controls VEGF receptor-2 (VEGFR-2) expression and suppresses VEGF-A signal in sprout stalks. The stalk cells follow the tip cell, have a high proliferation potential, stabilize sprouting vessels, and form the vascular lumen.3,4 In this way, the phenotype of the tip/stalk cell is controlled, but it is not known how the localized function is activated and regulated in tissues. To evaluate the functions of individual endothelial cells in tissues, a technology for analyzing positional heterogeneity in function is needed. To evaluate cell function in angiogenesis, analyzing gene expression at the mRNA level is very important. Previously, the relative mRNA expression between individual endothelial cells

was compared using in situ hybridization.5-12 Further, the analysis of tip/stalk cell markers using comprehensive gene expression has recently been reported. For example, genes enriched in tip cells were identified by isolating endothelial cells in Dll4+/- mouse mutants and the gene expression in tip cells and stalk cells was compared by collecting tip/stalk cells from sectioned mouse retina13 obtained using laser capture microdissection.14,15 However, these methods require cell dispersion or cell sectioning which could result in alteration in the original gene expression pattern. Therefore, new technologies for collecting the live tip/stalk cells selectively are required. Recently, a mouse ES cell-derived angiogenesis model has been widely used as a 3D model. The model has tip/stalk cells and reproduces cell-cell interactions in the living body. However, gene expression analysis in the model with the living state is very difficult because the model is constructed in hydrogel and the cells in the model cannot be collected. Previously, we developed DBCP which could collect the localized cells from tissue model on hydrogel16, but could not collect the localized cells in hydrogel. In this study, we achieved a collection of localized cells from the angiogenesis model in hydrogel. The expressions of the various marker genes from the endothelial cells in the living state were evaluated by improving the preparation of embryoid body (EB) to allow the approach of the DBCP to the model samples and optimizing a voltage to electrically puncture the cells.

1

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

■ MATERIALS AND METHODS

Cell culture Mouse ES cells (Strain129/SVE, passage11) were purchased from DS Pharma Biomedical Co., Ltd. The ES cells were cultured in Stem Medium (DS Pharma Biomedical Co., Ltd.) containing 0.1 mM β-mercaptoethanol (Millipore), 1% penicillin/streptomycin (Gibco), and 1,000 U/mL leukemia inhibitory factor (LIF, Millipore) to maintain the undifferentiated state. The medium was replaced every day. Embryoid body (EB) formation and differentiation At day 0, 150 cells were aggregated in hanging drops (20 µL) with differentiation medium and incubated in a humidified incubator (37°C in an atmosphere of 5 % CO2, 5 % O2). The differentiation medium consisted of Dulbecco’s modified Eagle’s medium (High Glucose, GlutaMAX™, Pyruvate; Gibco), 15 % FBS, 0.1 mM β-mercaptoethanol, 1 % penicillin/streptomycin, and 25 mM HEPES (Dojindo laboratories), 30 ng/mL vascular endothelial growth factor-A165 Mouse (mVEGF, Wako). After 4 days of culture (day 4), the EBs were transferred to 20 µL of 2.4 mg/mL collagen (Nitta Gelatin) drops, which consisted of Cell matrix I-A, Eagle’s MEM, and reconstitution buffer (8 : 1 : 1) on a 35-mm collagen coated dish. The culture dish containing the EB was turned upside-down and incubated for 20 min in 37°C to urge immobilization of EB near the surface of the collagen drop. After the addition of 2 mL differentiation medium, the EB was cultured at 37°C under hypoxia (5 % O2) for 6 days. The chart of differentiation culture is shown in Fig. S1a of the Supporting Information. The optical micrograph of the EB on day 4 and day 10

is shown in Fig. S1b.

Immunostaining of the EB-derived angiogenesis model At day 10, the EB in the collagen drop was fixed with 4 % paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and permeabilized using 0.1% triton X-100 (Wako) in PBS for 10 min. Subsequently, the sample was incubated in 1% bovine serum albumin (BSA; Wako) solution in PBS for 30 min at room temperature to prevent nonspecific binding of antibodies, and incubated with Alexa Fluor® 488 conjugated anti-mouse CD31 (1:1000, BioLegend) and antialpha smooth muscle actin (α-SMA) (1:500, Abcam) in PBS at 4°C for 90 min. Next, Alexa Fluor® 555-conjugated anti-rabbit IgG as a secondary antibody (1:1000; Abcam) was added to the sample and the sample was incubated at 4°C overnight. After each procedure was completed, the sample was washed three times in PBS. Samples were observed under a fluorescent microscope (Zeiss). The immunostaining pictures of the endothelial marker PECAM1 (CD31) and pericyte marker α-SMA are shown in

Fig. S1c.

Dissection of sprouting vessels using a surgical knife To confirm the differentiating states in sprouting vessels and the central core of the EB, each region was cut from the whole EB using a surgical knife (Feather blade handle; feather safety razor Co., Ltd) under a stereoscopic microscope (Leica)( Fig. S2a). The cut sprouting vessels and the remaining central core of the EB were collected using a syringe (Terumo) and transferred into a PCR tube containing 350 µL lysis buffer (Qiagen). The relative gene expression profiles of the sprout-

ing vessel and the central core of the EB are shown in Fig. S2b.

Page 2 of 5

Double-barrel carbon probe (DBCP) DBCP was fabricated, as described previously.16 First, to construct a file pipette, a quartz theta glass capillary (inner diameter, 0.9 mm; outer diameter, 1.2 mm; Sutter Instrument) was pulled using a CO2 laser puller (Sutter Instrument). Next, the pipette, which was pressurized using butane gas, was heated on a Bunsen burner to form the pyrolytic carbon layer inside the pipette. After the electric connection between the two barrels was confirmed by digital voltmeter, the extra carbon bonded to the tip of the pipette was removed by heating with the burner to separate the two barrels electrically. Finally, copper wires (φ 0.2 mm) were inserted into two barrels and fixed with a thermal shrinkage tube. DBCP was washed with 70 % ethanol (Wako) and dried at room temperature. The final outer diameter of DBCP was 20–30 µm. Collection of localized cells using DBCP To evaluate the change in local gene expression, a single cell or a few cells were collected using DBCP. First, the medium was removed from the dish and the dish was washed with 0.2 M sucrose five times. Subsequently, the solution was replaced with new 0.2 M sucrose. DBCP was set in oil-derived three-axis manual micromanipulator and positioned 3–5 µm away from the cells. An electrical pulse was applied at 500 V for 10 µs using an ECM 2001 Electro Cell Manipulator (BTX-Harvard Apparatus) under a phase contrast microscope on an anti-vibration table. After the target cells were punctured, the cell lysate was collected into DBCP using a syringe to apply a negative pressure inside DBCP and transferred into a PCR tube containing 80 µL lysis buffer and 5 µL carrier RNA (Qiagen). The schematic view of the cell collection system is shown in Fig. S3. Real-time PCR (qPCR) The cells were lysed, and total RNA was purified using an RNeasy Micro Kit (Qiagen). The RT reaction was performed with the QuantiTect reverse transcription kit (Qiagen). cDNA was synthesized as follows: 42°C for 2 min, 42°C for 30 min, and 95°C for 3 min on the peltier thermal cycler (Bio-RAD). qPCR with the sequence-specific primers was performed with the Light Cycler 1.5 System (Roche) based on the following conditions: 95°C for 10 min, 50 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 9 s. The primer sequences are shown as specific target amplification (STA) primer in Table S1 (Supplementary Materials). The forward and reverse primers for a pericyte marker (Acta2) were 5’-CCA ACC GGG AGA AAA TGA C-3’ and 5’-CAG TTG TAC GTC CAG AGG CAT A-3’, respectively. The expression levels were normalized to Gapdh levels. High-throughput PCR For comprehensive gene analysis, cDNA was pre-amplified using 2x TaqMan Preamp Master Mix (PN4391128, Applied Biosystems) and sequence-specific primers (STA primers in Table S1) as follows: 95°C for 10 min, 14 cycles of 95°C for 15 s and 60°C for 4 min. The pre-amplified cDNA was diluted 5-fold prior to analysis with Fast Start Taqman Gene Master Mix and EvaGreen DNA binding dye (Biotium) using the 48.48 Dynamic Arrays on a BioMark System (Fluidigm). The nested primer sequences are shown in Table S1. The expression levels were normalized to Gapdh levels.

■ RESULTS AND DISCUSSION

Influence of electric puncture To confirm that the electrical pulse by DBCP did not destroy the mRNA, MCF-7 single-cell samples were collected with and without electric punc-

2

ACS Paragon Plus Environment

Page 3 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

ture (Fig. S4a, b). MCF-7 is the cell line that adheres to the substrate weakly, so it could be aspirated using only negative pressure inside the DBCP. The DBCP was positioned 3–5 µm away from the cells and an electrical pulse was applied at 500 V for 10 µs between the two barrels. The averages and the standard deviations for the Gapdh copy number collected by electric puncture (500 V for 10 µs) and by aspiration were (3.7±1.4) x 103 and (2.4±2.0) x 103, respectively. In the box plot shown in Fig. S4c, the box and central bar indicated the lower/upper quartile and median, respectively. The whiskers showed minimum and maximum. There was no statistical difference between the two cellular collection methods. This result was in good agreement with the similar experiment done by our group with a smaller electric pulse (150 V for 10 µs). The conventional electric pulse was found to be not sufficient to electrically puncture the cells embedded in hydrogel. Therefore we optimized the electric pulse (500 V for 10 µs) for gene expression analysis during sprouting angiogenesis in the EB embedded in hydrogel. According to these results, the quantity of mRNA was hardly influenced by electric puncture and DBCP was shown to be applicable for analyzing single cell gene expression profiles.

Gene expression analysis in the top/middle region of sprouting vessels and the central core of the EB by DBCP Localized gene expression in the angiogenesis model was evaluated using DBCP, which allowed the collection of single to a few cells from the tissue model using electrical cell lysis with almost no destruction of the cellular mRNA. In this study, we applied DBCP to the tissue model in hydrogel for the first time. In the case that the EB was transferred to a collagen drop on the bottom of the culture dish, a thick hydrogel layer existed above the tissue model and prevented the approach of the DBCP to the model. Then the EB was formed on the cap of the collagen drop and turned upside-down after the EB was transferred. In this case, we immobilized the EB near the airhydrogel interface of the collagen drop. Using this method, the hydrogel existing above the tissue model became much thinner and the approach/puncture of the samples became easier. We collected the cells from the top/middle region of the sprouting vessel (Fig. 1a) and the surface of central core of the EB using DBCP. The phase contrast pictures before and after DBCP collection of the top region of the sprouting vessel is shown in Fig. 1b. We estimated the punctured area with Image J and confirmed that DBCP could puncture the cells within the range of 800 µm2. Judging from the Ct value of qPCR experiments, we estimated that DBCP can collect 1-100 cells in the top/middle region of sprouting vessels and 10-1000 cells in the central core of the EB. We think that the difference in collected cell number is because the cell density in central core of the EB is larger than in sprouting vessels. The relative gene expression profiles for each region are shown in Fig. 2. The expression of housekeeping gene (Actb) showed no difference between the sprouting vessel and the central core of the EB. The expression of an endothelial cell marker (Pecam1), a tip cell marker (Dll4), and a stalk cell marker (Robo4) in sprouting vessels was significantly higher than that in the central core of the EB. These results were well suited to the comprehensive gene expression profile shown in Fig. S2. This result implies that the cells in sprouting vessels differentiated into cells from the endothelial linage, including tip/stalk cells regardless of the position in the sprouting vessel. The expression of both Dll4 and Robo4 tended to be higher in the top region than in the middle region of the sprouting vessel,

although the expression of Pecam1 showed no difference (Fig. 2). The expression of a pericyte marker (Acta2) encoding α-SMA protein tended to be higher in the middle region than in the top region (Fig. 2). Previous studies reported that the expression of Dll4 was significantly higher in tip cells, 7,18,19 the expression of Robo4 was significantly higher in stalk cells,9 and the expression of Acta2 was significantly higher in pericytes.17 Therefore, our result implies that the tip/stalk cell is present at a relatively higher density in the top region than in the middle region and that the stabilization and maturation have been promoted in the middle region. Collectively, the degree of differentiation was different depending on the position of the sprouting vessel. As described above, we achieved the collection of the localized cells from the mouse ES cell-derived angiogenesis model using DBCP and investigated the positional difference in gene expression profiles due to the degree of differentiation. ■ CONCLUSION Here, we demonstrated the site-specific cell collection from a live tissue model in hydrogel using DBCP. The expressions of the various marker genes were evaluated by improving the preparation of EB and optimizing electric puncture condition. We found that vascular gene expression was much higher in sprouting vessels than in the central core of the EB because the cells in sprouting vessels might differentiate into cells from the endothelial linage, including tip/stalk cells. Vascular gene expression tends to be different between the top and middle regions within the sprouting vessel due to the difference in the degree of differentiation. Both tip and stalk cells were present in the top region in sprouting vessels and the cells in the middle region of the vessel became more mature. Collectively, these results show that DBCP is very useful for localized gene expression analysis to collect the localized cells from 3D live tissues in hydrogel while maintaining the tissue structure. This technique can be applied to comprehensive gene expression analyses in the medical field.

ASSOCIATED CONTENT SUPPORTING INFORMATION ADDITIONAL TABLE AND DATA AS NOTED IN THE TEXT. THIS MATERIAL IS AVAILABLE FREE OF CHARGE VIA THE INTERNET AT HTTP://PUBS.ACS.ORG

AUTHOR INFORMATION Corresponding Author *Corresponding author: Hitoshi Shiku, e-mail: [email protected] Tel & FAX: +81-22-795-6167 **Co-corresponding author: Tomokazu Matsue, e-mail: [email protected]

Author Contributions HI performed all experiments. HI, YN and HS designed the study. HI, YN, HS and TM wrote the paper. HI, YN and YZ performed high-throughput PCR. HY, YN, YZ, YT, KI, HS, and TM constructed ES cell-derived angiogenesis model, analyzed the results on gene expression profiles and approved the final version of the manuscript.

3

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 5

Present Address‖

Y. T.: School of Electrical and Computer Engineering, Kanazawa University, Kakuma-machi, Kanazawa 9201192, Japan Y. N.: Department of Microengineering,, Graduate School of Engineering, Kyoto University, Kyotodaigaku-katsura, Nishikyo-ku, Kyoto 615-8540, Japan Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was partly supported by Grants-in-Aid for Scientific Research (No. 25248032, No. 15H03542) and by the Cabinet Office, Government of Japan, through its “Funding Program for Next Generation World-Leading Researchers” (to HS). This work was supported in part by JST PREST (to YT). YN acknowledges the support received from Research Fellow of Japan Society for the Promotion of Science.

Fig. 1 Collection of the localized cells in the top/middle region of sprouting vessels and the central core of the EB by DBCP. (a)The image of the localized cell collection from the angiogenesis model by DBCP. (b) Representative phase contrast pictures before puncture (left) and after puncture (right) in the top region.

REFERENCES (1) Carmeliet, P.; Jain, R. K. Natrue 2000, 407, 249-257. (2) Hicklin, D. J.; Ellis, L. M. J. Clin. Oncol. 2005, 23, 1011-1027. (3) De Smet, F.; Segura, I.; Bock, K. D.; Hohensinner, P. J.; Carmeliet, P. Arterioscler Thromb. Vasc. Biol. 2009, 29, 639-649. (4) Blanco, R.; Gerhardt, H. Cold Spring Harb. Perspect. Med. 2013, 3, a006569. (5) Gerhardt, H. J. Cell Biol. 2003, 161, 1163-1177. (6) Jakobsson, L.; Franco, C. A.; Bentley, K.; Collins, R. T.; Ponsioen, B.; Aspalter, I. M.; Rosewell, I.; Busse, M.; Thurston, G.; Medvinsky, A.; Schulte-Merker, S.; Gerhardt, H. Nat. Cell Biol.y 2010, 12, 943-953. (7) Hellstrom, M.; Phng, L. K.; Hofmann, J. J.; Wallgard, E. ; Coultas, L.; Lindblom, P.; Alva, J.; Nilsson, A. K.; Karlsson, L.; Gaiano, N.; Yoon, K.; Rossant, J.; Iruela-Arispe, M. L.; Kalen, M.; Gerhardt, H.; Betsholtz, C. Nature 2007, 445, 776-780. (8) Benedito, R.; Roca, C.; Sorensen, I; Adams, S.; Gossler, A.; Fruttiger, M.; Adams, R. H. Cell 2009, 137, 1124-1135. (9) Phng, L. -K.; Gerhardt, H. Dev. Cell 2009, 16, 196-208. (10) Carmeliet, P.; De Smet, F.; Loges, S.; Mazzone, M. Nat. Rev. Clin. Oncol. 2009, 6, 315-326. (11) Phng, L. -K.; Potente, M.; Leslie, J. D.; Baddage, J.; Nyqvist, D.; Lobov, I.; Ondr, J. K.; Rao, S.; Lang, R. A.; Thurston, G.; Gerhardt, H. Dev. Cell 2009, 16, 70-82. (12) Strasser, G. A.; Kaminker, J. S.; Tessier-Lavine, M. Blood 2010, 115, 5102-5110. (13) Toro, R. D.; Prahst, C.; Mathivet, T.; Siegfield, G.; Kaminker, J. S.; Larrivee, B.; Breant, C.; Duarte, A; Takakura, N.; Fukamizu, A.; Penninger, J.; Eichmann, A. Blood 2010, 116, 4025-4033. (14) EmmertBuck, M. R.; Bonner, R. F.; Smith, P. D.; Chuaqui, R. F.; Zhuang, Z. P.; Goldstain, S. R.; Weiss, R. A.; Liotta, L. A. Science 1996, 274, 998-1001. (15) Schutze, K.; Lahr, G. Nat. Biotechnol. 1998, 16, 737-742. (16) Nashimoto, Y.; Takahashi, Y.; Takano, R.; Miyashita, K.; Yamada, S.; Ino, K.; Shiku, H.; Matsue, T. Anal. Bioanal. Chem. 2014, 406, 275-282. (17) Jakobsson, L.; Kreuger, J.; Holmborn, K.; Lundin, L.; Eriksson, I.; Kjellen, L.; Claesson-Welsh. Dev. Cell 2006, 10, 625-634. (18) Siekmann, A. F.; Lawson, N. D. Nature 2007, 445, 781-784. (19) Suchting, S.; Freitas, C.; le Noble, F.; Benedito, R.; Breant, C.; Duarte, A.; Eichmann, A. Proc. Natl. Acad. Sci. USA 2007, 104, 3225-3230

Fig. 2 Gene expression analysis of the cells collected from the top/middle region of sprouting vessels and the central core of the EB. (P* < 0.05, P**< 0.01, P***< 0.005)

4

ACS Paragon Plus Environment

Page 5 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

For TOC

5

ACS Paragon Plus Environment