Mxpro software version 4

Total RNA was extracted from spheroids using TRI-reagent (Sigma-Aldrich, Merck, MO, USA); a RNeasy mini kit (Qiagen, Hilden, Germany) was utilised according to the manufacturer’s protocol with an additional on-column DNase digestion step (RNase-Free DNase Set; Qiagen, Hilden, Germany). Complementary DNA synthesis was synthesised from 2 µg RNA, using the RNA-dependent DNA polymerase, Moloney murine leukaemia virus reverse transcriptase (Promega WI, USA). The samples were diluted to a final concentration of 10 ng/µL. Following optimisation of primers and ensuring the annealing temperature provided ~100% amplification efficiency per cycle (data not shown), qPCR was performed, as previously described,⁴⁷ using SYBR Green PCR mix (Qiagen, Hilden, Germany) and the following Qiagen QuantiTect primers, LGR5 (cat. no. QT00027720) and CD133 (cat. no. QT00075586), with gene expression normalised interchangeably with both housekeeping genes TATA-binding protein (TBP; cat. no. QT00000721) or hypoxanthine phosphoribosyl transferase (HPRT; cat. no. QT00059066). Amplification data were analysed using MxPro software version 4.10 (Agilent Technologies, CA, USA).

RNA was extracted using the RNeasy micro kit (QIAGEN, Hilden, Germany) and cDNA was synthesized using the reverse transcription using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan), according to the manufacturer’s instructions. Real-time PCR assay was carried out using 2 μl of cDNA as the template and 10 μl of Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on the Stratagene Mx 3005P QPCR System (Agilent Technologies, Santa Clara, CA, USA).
The primers are listed in Supplementary Table 3. m-CSF1 and s-CSF1 were distinguished by specific primers (19 (link)). The data was analyzed based on 2^−ΔΔCt method and normalized by GADPH expression using the MxPro software version 4.10 (Agilent Technologies). The thermal cycling conditions for the PCR were 95°C for 10 min for polymerase activation, followed by 45 cycles of 95°C for 15 s for denaturation, and 60°C for 1 min for extension.