Anti cd133 2 pe

Primary gliostoma cells were dissociated from cell culture dishes and resuspended in PBS with 0.5% BSA and 2mM EDTA and incubated with CD133/1 microbeads (Miltenyi Biotech) followed by positive magnetic cell separation using several MACS columns. The purified CD133⁺ cells were stained with anti- CD133/2-PE (Miltenyi Biotech) and analyzed on a BD FACSCalibur. Then, neurosphere cultures were obtained by growing CD133⁺ tumor cells in stem cell-permissive DMEM/F12 medium supplemented with 20ng/ml each of human recombinant epidermal growth factor and human recombinant basic fibroblast growth factor (R and D Systems), and human leukemia inhibitory factor (Chemicon) and 2% B27 (Life Technologies). These culture conditions enabled tumor cells to retain the molecular characteristics of the primary tumor, with only minor changes in differentiation, expression pattern, and genetic mutation profile.
Cultured primary astrocytes were generated from a slightly injured brain tissue fragment obtained after consent from a patient with cerebral trauma. The grey matter was dissected and dispersed repeatedly after washing in PBS. Primary astrocytes were cultured according to the protocol described by Darling et al [32].

Anti-CD34-PE, anti-CD15-PE, anti-CD184-PE, anti-SSEA4-V450 and the isotype controls were purchased from BD Biosciences. Anti-CD133/2-PE was from Miltenyi Biotec. Anti-Sox2 was from Millipore. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT), dimethylsulfoxide (DMSO), 4-6-diamino-2-phenylindole (DAPI), cis-diammineplatinum-II-dichloride, doxorubicin hydrochloride, methotrexate hydrate and Fluoromount Aqueous Mounting Medium were from Sigma Aldrich. TrypLE and ACK lysing buffer were from Life Technologies. EnVision™ FLEX, High pH and EnVision FLEX Target Retrieval Solution, High pH were obtained from Dako. Protease inhibitor cocktail tablets were from Roche. SuperSignal West Pico Chemoluminescent Substrate was from Thermo Scientific.Bergisch Gladbach, Germany.

Cell viability and stem cell surface marker expression were analysed by flow cytometry 0 h, 18 h, and 24 h after isolation. For staining, samples were treated with the following antibodies: anti-CD34-FITC (clone: AC136), anti-CD133/2-PE (clone: 293C3), isotype control mouse IgG 2b-PE (Miltenyi Biotec GmbH), anti-CD45-APC-H7 (clone: 2D1), and 7-AAD (BD Biosciences, Germany). To reduce unspecific bindings, FcR blocking reagent (Miltenyi Biotec GmbH) was added. After incubation for 10 min at 4°C, samples were measured with LSR-II flow cytometer (BD Biosciences) and data analysis was realised with FACSDiva software (BD Bioscience). For evaluation, Boolean gating strategy was arranged on the ISHAGE guidelines for CD34⁺ cell analysis [48 ] in following order:

Step  1: selection of cell population (Figure S1A, in Supplementary Material available online at http://dx.doi.org/10.1155/2016/7152761).

Step  2: selection of CD45⁺ cells (Figure S1B).

Step  3: selection of viable CD45⁺ cells (Figure S1C).

Step  4: selection of viable CD45⁺/CD34⁺ cells (Figure S1D).

Step  5: selection of viable CD45⁺/CD34⁺/CD133⁺ cells (Figure S1E).

Calculation of cell viability and surface marker integrity was based on the following equations: $\begin{array}{c}\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)=\frac{{\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\hspace{0.17em}\hspace{0.17em}\mathrm{C}\mathrm{D}\mathrm{45}}^{+}\hspace{0.17em}\hspace{0.17em}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{{\mathrm{C}\mathrm{D}\mathrm{45}}^{+}\hspace{0.17em}\hspace{0.17em}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\times \mathrm{100},\\ \mathrm{S}\mathrm{u}\mathrm{r}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{e}\hspace{0.17em}\hspace{0.17em}\mathrm{m}\mathrm{a}\mathrm{r}\mathrm{k}\mathrm{e}\mathrm{r}\hspace{0.17em}\hspace{0.17em}\mathrm{p}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{n}\left(\%\right)\\ =\frac{{\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\hspace{0.17em}\hspace{0.17em}\mathrm{C}\mathrm{D}\mathrm{45}}^{+}/{\mathrm{C}\mathrm{D}\mathrm{34}}^{+}/{\mathrm{C}\mathrm{D}\mathrm{133}}^{+}\hspace{0.17em}\hspace{0.17em}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{{\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\hspace{0.17em}\hspace{0.17em}\mathrm{C}\mathrm{D}\mathrm{45}}^{+}\hspace{0.17em}\hspace{0.17em}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\times \mathrm{100}.\end{array}$