Rat anti mouse cd11b

The cell surface markers of MSCs were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, cells that reached 90% confluence were harvested using 0.25% EDTA and washed twice in Dulbecco’s phosphate buffered saline supplemented with 10% FBS. The cells for detecting CD11b, CD34, CD45, Sca-1, CD44 and CD73 were labeled directly with BB515 or PE-conjugated CD markers (rat anti-mouse CD11b [1: 100, BD Pharmingen; BD Biosciences, Franklin Lake, NJ, USA], rat anti-mouse CD34 [1: 100, BD Pharmingen], rat anti-mouse CD45 [1: 100, BD Pharmingen], rat anti-mouse Sca-1 [1: 100, BD Pharmingen], rat anti-mouse CD44 [1: 100, BD Pharmingen], rat anti-mouse CD73 [1: 100, BD Pharmingen]).

For macrophage surface marker analysis, macrophages were incubated with fluorescent labeled rat anti-mouse CD11b, iNOS, and CD206 antibodies (BD Biosciences Pharmingen, San Diego, CA).
To measure macrophage infiltration in the tumor sites and blood, the tumor cells and peripheral cells were stained with Alexa Fluor^® 594-conjugated rabbit anti-mouse CD206 (Abcam, Cambridge, MA) and FITC-labeled anti-mouse F4/80 (Abcam, Cambridge, MA).

To prepare the anti-CD11b microbeads, 25 μl of sheep anti-rat Dynabeads (Invitrogen™) were washed three times with 0.75 ml of RPMI 1640 medium using a magnetic scaffold, allowing 1 min for the beads to settle each time. The Dynabeads were then suspended in 0.5 ml of serum-free 1640 medium and incubated overnight at 4 °C on a rotator (continuous rotation at 8 RPM) with 8 μl of rat anti-mouse CD11b (BD Pharmingen™, cat. no. 553308). After incubation, the anti-CD11b-conjugated microbeads were washed twice, resuspended in 0.5 ml of 1640 medium, and mixed with 0.5 ml prepared mouse brain cells. The mixture was incubated on a rotator for 30 min at 4 °C and then placed on a magnetic scaffold for 1 min, after which the supernatant was removed. The resulting MG-bead mixtures from six mice were pooled as one sample and washed with PBS before being used for RNA purification.

The freshly excised eyeballs were directly embedded in optimal cutting temperature (OCT) compound (Fisher Scientific, Pittsburgh, PA, USA) in preparation for sectioning. 14 μm-thick cryosections were made from frozen blocks (Leica CM3050 S; Leica Biosystems, Buffalo Grove, IL, USA). Following the fixation of sections with 4% PFA for 15 min at room temperature, tissue sections were rinsed by 0.3% Triton X-100 in PBS and blocked with blocking buffer (1× PBS/1% BSA/0.3% Triton X-100) for 1 h. Slides were stained overnight at 4°C with primary antibodies. The primary antibodies used were rat anti-F4/80 (1:400, NB600-404; Novus), rat anti-mouse CD11b (1:50, #550282; BD Pharmingen), rabbit anti-vimentin (1:100, #5741; Cell Signaling Technology), and donkey anti-human IgG (H+L) conjugated with Alexa Fluor 488 that binds to the heavy (H) and light (L) chains of humanized IgG (1:400, #144222; Jackson ImmunoResearch Laboratories), which were all diluted in PBS with 0.3% Triton X-100 and 5% BSA. The secondary antibodies with 4′,6-diamidino-2-phenylindole (DAPI; #9542; Sigma-Aldrich) counterstain used were goat anti-rat IgG-Alexa Fluor 594 and goat anti-rabbit IgG-Alexa Fluor 594. Fluorescence images were acquired by a Leica DM6 microscope. Image analysis was performed with Adobe Photoshop software. CD11b⁺ or F4/80⁺ cells were detected and counted by using ImageJ software.

A total of 2 × 10⁵ of transduced cells/ml were incubated for 7 days in RPMI 1640 GlutaMAX supplemented with 10% FCS, 10 ng/ml hIL-6, 5 ng/ml IL-3, 5 ng/ml GM-CSF, and 10 ng/ml G-CSF. The medium was exchanged every second day. On day 7, the medium was changed to RPMI 1640 GlutaMAX supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 ng/ml G-CSF. The medium was exchanged every second day until day 11. On day 11, cells were analyzed by FACS using the following antibody: rat anti-mouse Gr-1 (BD 553128) and rat anti-mouse CD11b (BD 553312) on FACSCanto II.