The study of phospholipids in single cells using an integrated microfluidic device

Transcription

1 Supporting Information: The study of phospholipids in single cells using an integrated microfluidic device combined with matrix-assisted laser desorption/ionization mass spectrometry Weiyi Xie,, Dan Gao, *,, Feng Jin, Yuyang Jiang,, Hongxia Liu,, Department of Chemistry, Tsinghua University, Beijing , China State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen , China Key Laboratory of Metabolomics at Shenzhen, Shenzhen , China Neptunus Pharmaceutical Technology Center, Shenzhen , China School of Medicine, Tsinghua University, Beijing , China Corresponding authors. Phone: S-1

2 Reagents and Materials Negative photoresist (SU and 2007) and developer were purchased from MicroChem (Newton, MA, USA). Polydimethylsiloxane (PDMS) and curing agent were purchased from Dow Corning (Midland, MI, USA). 9-aminoacridine (9-AA) was obtained from Sigma-Aldrich (St. Louis, MO, USA). α-cyano-4-hydroxy cinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), Peptide Calibration Standard II and indium tin oxide (ITO) coated glass slide were purchased from Bruker Daltonics GmbH (Leipzig, Germany). F-12K culture medium was purchased from Genom Biotech Co., Ltd. (Hangzhou, China). Fetal Bovine Serum (FBS) was obtained from Biochrom Co., Ltd. (Cambridge, UK). Calcein AM and ethidium homodimer-1 (Live/Dead assay kit) were acquired from Invitrogen Life Technologies (Carlsbad, CA, USA). LC-MS-grade methanol, ethanol and AR-grade chloroform were purchased from Merck Chemical Co. (Darmstadt, Germany). A549 cell line was purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). S-2

3 The Lipid Extraction Experiment The A549 cells were firstly released from the petri dishes by trypsinization with 0.25% Trypsin EDTA (Gibco). Then, the cell suspension containing A549 cells was centrifuged at 800 rpm for 5 min, and then the supernatant was removed. Next, 400 µl of a mixed chloroform-methanol (1:3, v/v) solvent was added into the tube and vortex-mixed for 3 min. The tube was centrifuged at 4000 rpm for 5 min, and the supernatant was collected, followed by mixing with 200 µl of chloroform and 100 µl of water. After vortex mixing for 3 min and centrifuged at rpm for 5 min, the organic phase was collected for further analysis. S-3

4 Figure S1. Photo of the fabricated microfluidic chip. S-4

5 Figure S2. The formation of cell array using different concentrations of cells on the microfluidic chip with 40 µm microwell array. 10 µl of cell suspension was introduced into the chip. Scale bar: 100 µm. S-5

6 Figure S3. MALDI mass spectra of a single cell with 9-AA as matrix in positive and negative ion mode. (A) Mass spectrum of 9-AA in positive ion mode. (B) Mass spectrum of a single cell using 9-AA as matrix in positive ion mode. (C)Mass spectrum of 9-AA in negative ion mode. (D) Mass spectrum of a single cell using 9-AA as matrix in negative ion mode. S-6

7 Figure S4. MS/MS spectra of the detected phospholipids. S-7

8 Figure S5. The deposition of 9-AA solution on cell array by two methods: (A) 9-AA solution was sprayed by an automatic sprayer; (B) 9-AA solution was pipetted manually. Scale bar: 100 µm. S-8

9 Figure S6. (A) The fully desorption and partial desorption region shot by the laser beam with a small focus setting smartbeam II laser. Scale bar: 100 µm. (B) The micrograph of 1 cell, 2 cells and 3 cells in the single cells array, respectively. Scale bar: 25 µm. S-9

10 Considering that the mass spectrometry signals of phospholipids from single cells might be influenced by the background interference of the matrix, we conducted an experiment on A549 cells without any matrices. ITO coated glass slide was used as substrate. The result was shown in Figure S7. It is obvious that components in cells cannot be ionized without matrix, which indicated that matrix is necessary in MALDI measurement of cells using ITO coated glass slide. Figure S7. The MALDI mass spectrum of A549 cells with 9-AA as matrix (blue) and A549 cells alone (red). S-10

11 Figure S8. MALDI image of 9-AA with selected ion at m/z = 195 in a region of interest (ROI). S-11

12 Figure S9. (A) MALDI-MSI spectrum of a 9-AA deposited area on ITO coated glass slide without cells array. (B) MALDI image of m/z 782. Scale bar: 100 µm. S-12

13 Figure S10. The mass spectrometry signal intensity of PC(34:1) in one, two or three cells, as well as the linear correlation analysis. Data shown are averages plus and minus the standard deviation of ten independent experiments. S-13

14 Figure S11. MALDI image and data extraction of PC(34:1) and PC(36:2). (A) The measured area and MALDI image of PC(34:1) and PC(36:2), respectively. (B and C) Phospholipid intensity extraction from pixels in MALDI image. Yellow character stands for pixel number and white character stands for cell counts in every pixel. S-14