Raman Spectroscopy Analysis of Cell Cultures

The Raman spectra were measured by a Renishaw in Via Raman spectrometer (controlled by WiRE 3.4 software, Renishaw, UK) connected to a Leica microscope (Leica DMLM, Leica Microsystems, Buffalo Grove, IL, USA), equipped with a 785 nm near-IR laser that was focused through a 63 × NA = 0.90 water immersion objective (Leica Microsystems, USA). The laser intensity before and after travelling through the 50× objective was 110 mW and 29.4 mW, respectively (measured with LaserCheck, Coherent Inc., Portland, OR, USA). The standard calibration peak for the spectrometer with silicon mode at a static spectrum was 520.5 ± 0.1 cm^-1. Cells cultured with blank media, or media with G-CSF for 10 days and 7 weeks were seeded on MgF2 (United Crystals Co., Port Washington, NY, USA) for 24h prior to Raman spectra collection. The exposure time was 10 s for one accumulation at 100% laser power for all of the cell samples. A total of 50 spectra (randomly 5 spots for 5 spectra per cell) were acquired for each group in the fingerprint region (600 −1800 cm⁻¹) which has been proven effective in evaluation of molecular changes of cancer cells ^{13 (link), 21 (link)–24 (link)}