Data on step-by-step atomic force microscopy monitoring of changes occurring in single melanoma cells undergoing ToF SIMS specialized sample preparation protocol

Data included in this article are associated with the research article entitled ‘Protocol of single cells preparation for time-of-flight secondary ion mass spectrometry’ (Bobrowska et al., 2016 in press) [1]. This data file contains topography images of single melanoma cells recorded using atomic force microscopy (AFM). Single cells cultured on glass surface were subjected to the proposed sample preparation protocol applied to prepare biological samples for time-of-flight secondary ion mass spectrometry (ToF SIMS) measurements. AFM images were collected step-by-step for the single cell, after each step of the proposed preparation protocol. It consists of four main parts: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying. In total 13 steps are required, starting from imaging of a living cell in a culture medium and ending up at images of a dried cell in the air. The protocol was applied to melanoma cells from two cell lines, namely, WM115 melanoma cells originated from primary melanoma site and WM266-4 ones being the metastasis of WM115 cells to skin.


Specifications
Physics, Chemistry, Biology More specific subject area

Biophysics, Biochemistry
Type of data Figures How data was acquired Atomic force microscope (AFM): XE120 Park Systems. Surface images (topography and deflection modes and 3D representation of topography) recorded by Xe120 system (saved originally in tiff format) Data format Analyzed Experimental factors Living melanoma cells were cultured on the glass coverslips, placed in a Petri dish, and immersed in a culture medium. Such samples were further used for the AFM measurements. Experimental features AFM images of surface topography of single melanoma cells were measured after each step of the applied preparation protocol. The AFM topography was acquired for the same cell.

Data accessibility
Data are provided with this article

Value of the data
The data presented here essentially show changes on a cell surface upon application of the ToF SIMS specific sample preparation protocol.
Recorded images enable to estimate the effect of fixation, salt removal, dehydration, and air-drying on surface topography and morphology of single cells.
The proposed protocol for sample preparation can be applied to prepare single cells cultured on bare silicon surface for ToF SIMS measurements delivering mass spectra of single cells.

Data
The data presented below, show an exemplary sequence of the surface topography and deflection ("error") images (a & b) recorded using an atomic force microscope (model: Xe120, from Park Systems, Korea) for a single cell after each step of the applied ToF SIMS specific sample preparation protocol, accompanied by a 3D representation of a cell surface (c). The protocol involve four main stages, i.e. fixation, salt removal, dehydration and air-drying. The recorded data show changes on the The AFM images presented below, show surface changes of single WM266-4 cells acquired after each step of the ToF SIMS specialized sample preparation protocol. The presented protocol enables to record mass spectra (ToF SIMS) on single cells [1,2] (Figs. 2.1-2.13).

Cell culture
WM115 (skin primary tumor site) and WM266-4 (metastasis to skin) melanoma cells were cultured in the RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). The cells were grown in 25 cm 2 culture flasks (Sarstedt) in the incubator (NuAire) (37°C and a 95% air/5% CO 2 atmosphere). After several passages (6-8), cells were seeded on clean and sterile glass coverslips or silicon substrates, placed in the Petri dishes (Sarstedt), and further cultured for 48 h in the corresponding media and culture conditions. For the AFM topography measurements cells were cultured directly on a bare glass coverslips while for ToF SIMS experiments they were grown on bare silicon substrates.

ToF SIMS specific protocol
The protocol for single cell preparation for ToF SIMS experiments follows through the following steps (step number correlates with image number): 1. Living cell cultured on bare silicon surface (for AFM measurementson glass surface). 2. Fixation with paraformaldehyde. 3. Rinsing with 50% phosphate buffered saline solution (PBS). 4. Rinsing with 25% PBS.

Atomic force microscopy
Topography of cells' surface was recorded after each step of the sample preparation protocol using a commercial AFM set-up working in contact mode (model Xe120, Park Systems, Korea). Images were obtained using V-shaped silicon nitride cantilevers, characterized by nominal spring constant of 0.03 N/m (PNP-TR-customized, Nanoworld). The set point ranged from 0.2 nN to 0.8 nN (adjusted during the topography acquisition) while the scan rate range was set between 0.3 Hz and 1 Hz depending on the scan size. In the presented measurements, glass coverslips with living cells, placed   in the Petri dish and immersed in a culture medium (RPMI-1640 culture media supplemented with 1% HEPES (pH 7.5)), were mounted on the top of a piezoelectric scanner. First, an image of a single alive cell was recorded in the culture medium. Then, images of the same cell were collected after each   steps of the proposed protocol for single cells' preparation for ToF SIMS spectroscopy. In total, a sequence of thirteen images was collected. Images were analyzed using XEI (dedicated AFM software provided by Park Systems) and WSxM 5.0 software [3], only the contrast and slope of the images were adjusted when necessary.