Fcr blocking buffer

In order to verify the difference of FOXO1 gene expression in different cells of MDS, BMNC from 5mL fresh bone marrow samples of 3 matched MDS patients were isolated. After cultured at 37 °C and 5% CO₂ saturated humidity, BMNC in logarithmic growth phase were taken for subsequent cell isolation. According to the manufacturer’s instructions, CD3+ and CD34+ cells were isolated using CD3 and CD34 immunomagnetic beads (Miltenyi Biotec, 130-050-101, 130-046-702) respectively. According to the manufacture’s protocol, for positive selection, cell pellet was resuspended in 300 μL MACS buffer (Miltenyi Biotec, 130-091-222) and 1 × 10⁸ total cells were incubated with 100 μL of FcR blocking buffer (Miltenyi Biotec, 130-100-453). CD3 or CD34 microbeads were incubated together with cell pellet for 30 min in the refrigerator (2–8 °C). Cells were added and washed by 10 mL of MACS buffer, then centrifuged at 300× g for 10 min. After discarding the supernatant and resuspend the cells at 500 μL buffer, and sort CD3 + or CD34 + cells by LS magnetic columns (Miltenyi biotec, 130-042-401). The sorted cells were detected by flow cytometry, and the positive expression rate reached more than 90%. BMNC of three MDS patients were sorted before and after disease progression for subsequent RT-PCR detection.

Isolated microglia from MS donors (n = 7) were incubated for 15 min in FcR-blocking buffer (1:5; Miltenyi Biotec), to block unspecific binding of antibodies to Fc-receptors. Next, microglia were incubated with conjugated primary antibodies (Additional file 1: Supplementary Table 5) diluted in beads buffer (0.5% BSA and 2 mM EDTA in PBS, pH 7.6) for 30 min at 4 °C. To determine viability, cells were incubated with viability dye efluor506 (Additional file 1: Supplementary Table 5).
To assess minimal phenotyping of isolated microglia, CD45 and CD11b expression was determined. In addition, expression of homeostatic microglia receptors, P2Y₁₂, CX3CR1 and GPR56 was measured. To exclude infiltrating leukocytes in the samples collected from MS lesion tissue, CD3, CD19, CD56 and CD66b were included.
Surface protein expression was detected on a 3-laser BD FACSCanto II machine (BD Biosciences) with software BD DIVA version 8.1. FlowJo software version 10.1 (Ashland, OR, USA) was used to determine median fluorescence intensity.

ES cells (1 × 10⁸) were incubated with 300 µL of CD133 microbeads and 100 µL of FcR blocking buffer (Miltenyi Biotech). CD133-positive cells were isolated using LS columns (Miltenyi Biotech). CD133-negative cells were depleted of labelled (CD133-positive) cells by passing the cells through two LD columns (Miltenyi Biotech). Cell surface expression of CD133 was confirmed by flow cytometry immediately after separation: >90% of the CD133-positive selection expressed CD133, and CD133 was expressed by <5% of the CD133-negative selected cells. CD133-positive A673 and TC-32 cells remained positive over 15 passages.