Anti α6 integrin pe goh3

Testicular single-cell suspensions were prepared from 2–3-month-old Col1a1-4F2A-OSKM mice as described previously [34 ]. The immunomagnetic selection of α6⁺ cells was performed using anti-α6 integrin-PE (GoH3) (BD Pharmingen) and anti-PE microbeads (Miltenyi Biotec) according to the manufacturer's protocol. Hoechst staining (5 μg/ml) of the cell suspensions was performed as described previously [34 ]. Cells were then labelled with β2m-FITC (Santa Cruz) and anti-CD117-APC (2B8) antibodies (BD Pharmingen). Propidium iodide (Sigma) was added before cell sorting to exclude dead cells. To analyse the transcription factors, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Pharmingen) and stained with the following antibodies: Alexa Fluor 488 mouse anti-Nanog (BD Pharmingen 560261 clone M55-312), Alexa Fluor 647 mouse anti-Sox2 (BD Pharmingen 560294 Clone 245610), rabbit anti-c-Myc (Cell Signaling), rabbit anti-Kfl4 (Abcam), rabbit anti-Lin28 (Cell Signaling), rabbit IgG isotype control (Cell Signaling) and secondary anti-rabbit Alexa Fluor 488 antibody along with APC-anti-PLZF antibody (R&D). Analyses and cell sorting were performed on ARIA, LSR II and FACSCalibur flow cytometers (Becton Dickinson).

Testis cells from 2-month-old B6 mice were obtained as described previously [40 (link)]. The testis albuginea was removed and seminiferous tubules were dissociated using enzymatic digestion by collagenase type I at 100 u/mL for 15 min at 32 °C in HBSS supplemented with 20 mM HEPES pH 7.2, 1.2 mM MgSO₄ 7H₂O, 1.3 mM CaCl₂ 2H₂O, 6.6 mM sodium pyruvate, and 0.05% lactate. After an HBSS wash and centrifugation, the pelleted tubules were further incubated in cell dissociation buffer (Invitrogen) for 25 min at 32 °C. The resulting whole cell suspension was successively filtered through a 40 μm then 20 µm nylon mesh to remove cell clumps. After an HBSS wash, the cell pellet was resuspended in incubation buffer (HBSS supplemented with 20 mM HEPES pH 7.2, 1.2 mM MgSO₄ 7H₂O, 1.3 mM CaCl₂ 2H₂O, 6.6 mM sodium pyruvate, 0.05% lactate, glutamine and 1% fetal calf serum) and stained with Hoechst 33,342 (5 µg/mL) for 1 h at 32 °C in a water bath. Cells were then labeled with monoclonal antibodies (1 µg per 10⁶ cells) from BD Pharmingen: anti-c-Kit-biotin (2B8), and anti-α-6 integrin-PE (GoH3). The cell sorting was performed on ARIA (Becton Dickinson).