Evos floid cell imaging station fluorescent microscope

To evaluate stem cell marker expression, neurospheres were dissociated mechanically or enzymatically with Accutase (Gemini Bioscience, Sacramento, CA). To facilitate adherence, cells were plated on poly-L-lysine/laminin coated four-well plates in neurosphere media. Cells were fixed in 4% paraformaldehyde, blocked and permeabilized with a 5% bovine serum albumin (BSA) with 0.6% Triton-× 100 and then treated with the primary antibodies Nestin (Abcam, Cambridge, MA), Sox2, Musashi 1, CD44, Bmi-1 (Cell Signaling Technology, Danvers, MA), CD133 (Biorbyt, Cambridge, UK) and A2B5 (A2B5 clone 105, ATCC, Manassas, VA). A “no primary control” was included for all antibodies tested for all cell lines. For these, the cells were incubated with only the antibody diluent (2.5% BSA, 0.3% triton, balance PBS). Cells were then treated with a fluorochrome-conjugated secondary antibody followed by Prolong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific, Waltham, MA). Samples were examined under an EVOS FLoid Cell Imaging Station fluorescent microscope (Thermo Fisher Scientific, Waltham, MA).

Cells were cultured on 4-well plates (Nunc), fixed in 4% paraformaldehyde, washed, blocked and permeabilized with a 5% bovine serum albumin (BSA) with 0.6% Triton-× 100 and then treated with the primary antibodies NAMPT and NAPRT (GeneTex). For analysis of stem cell markers in NB1691-sp cells, cells were plated on laminin/poly-L-Lysine (Sigma-Aldrich) coated plates and treated with Bmi1 or Musashi primary antibodies (Cell Signaling Technology). Cells were then treated with Alexa Fluor 594 -conjugated secondary followed by Prolong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific). Samples were examined under an EVOS FLoid Cell Imaging Station fluorescent microscope (Thermo Fisher Scientific).

Curcumin-induced ROS was visualized and quantitated using the general oxidative stress indicator CM-H2DCFDA (Thermo Fisher Scientific, Waltham, MA). CM-H2DCFDA passively diffuses into cells and reacts with ROS to yield a fluorescent adduct. For quantification, cells were split into 96-well plates in cell culture media with the addition of 5% FBS to cause adherence to the well bottoms. Samples were treated with 25 μM of curcumin in phenol red free media for 30 min, 4 h, and 24 h. Cells were incubated with 0.5 μM CM-H2DCFDA in PBS for 5 min subsequently washed in PBS and read at an excitation of 495 nm and an emission of 525 nm using BoiTek Synergy HT plate reader. Data is presented as fold change from non-treated cells. Curcumin-induced ROS activity was also examined using fluorescent microscopy. Dissociated GSCs were plated in neurosphere media on poly-L-lysine/laminin coated four-well plates. CM-H2DCFDA fluorescence was evaluated at 1, 6 and 24 h post curcumin (25 μM) treatment. Images were obtained using the EVOS FLoid Cell Imaging Station fluorescent microscope (Thermo Fisher Scientific, Waltham, MA).