Facs aria 3 apparatus

In order to quantify the percentage of the circulating cancer cells in the blood obtained from NOD/SCID mice treated with vehicle, olaparib, EZRi, olaparib combined with EZRi, fresh whole blood was taken from hearts of deeply anaesthetized mice by cardiac puncture. In brief, to minimize hair flying around, 70% ethanol solution was used to wet down and disinfect the fur. 21‐gauge needles, 1 mL syringes and blood collection microtubes were recoated with fresh 0.5 % w/v heparin for anticoagulation. 250 μL whole blood sample in each group was used and red blood cells were lysed by RBC Lysis Buffer (Biolegend) according to the user manual. mCherry positive cancer cells in each group were sorted via a FACS Aria III apparatus (BD Biosciences) 35 (link), 36 (link).

For flow cytometry analysis of cellular components in the injured brain, the isolated cells were stained with fluorescently labeled antibodies: CD45-FITC, CD11b-APC, Ly6G-PE, CD3-PE-Cy7, CD45R (B220)-APC, NK1.1-PE, CD206-PE, CD86-PE-Cy7 and the appropriate isotype control according to the manufacturer’s protocols (eBioscience). To detect Tregs in the brain and spleen, the isolated cells were stained using a commercial Mouse Tregs Staining Kit (eBioscience). Briefly, cells were surface stained with anti-mouse CD4-FITC and anti-mouse CD25-PE for 30 min at 4 °C, fixed, and then permeabilized overnight at 4 °C using the fixation/permeabilization solution and subsequently stained with the anti-mouse/rat Foxp3-APC antibody. Flow cytometry was performed on a FACS Aria III apparatus (BD Bioscience) and the obtained data were analyzed by Flow Jo software 7.6.1(Tree Star, US).

RNA was isolated from DAPI¯/ CD11b⁺/ F4/80⁺ phenotype cells sorted using FACS AriaIII apparatus (BD). Total RNA samples were prepared using the Total RNA Mini Plus kit (A&A Biotechnology, Gdynia, Poland), according to manufacturer’s procedure. cDNA was synthesized with M-MuLV reverse transcriptase and random hexamers (EURx, Gdańsk, Poland). Starters characteristic for M1 and M2-specific cytokines and chemokines genes were employed (mArg-1, mCCL-22, mMRC-1, mCCL-17, mCSF-1, mMMP-9, mIL-10, mCXCL-11, miNOS, mIL-1b, mCXCL-9, mIL-12a, acc. to Huang et al. [36 (link)]; mVEGFa, mPlGF [57 (link)]; mVEGFc and mNRP-1 [58 (link)]). mRNA levels’ measurements were performed by SG qPCR Master Mix (2x) (EURx, Gdańsk, Poland). The amplification was performed using a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Relative quantitation of mRNA was performed using ΔΔC_T method with β-actin as a reference gene. Means were calculated from three independent repeats.