Fluoreporter lacz flow cytometry kit

FACS purification procedures were as previously described^{49 (link)}. Bu-HFSCs were sorted on the basis of surface expression of integrin α6 and CD34 and/or cytosolic YFP or RFP. Cells expressing bacterial β-galactosidase were purified using the FluoReporter lacZ Flow Cytometry kit (Molecular Probes). Hair germ cells were purified on the basis of the expression of YFP and integrin α6, but negative for CD34 and Sca1 markers. Cells were collected into either Trizol LS reagent (Invitrogen) for RNA purification, or culture media for colony formation, or pre-coated 15-ml Flacon tubes for ChIP-seq and protein purification.

Skulls were fixed in 4% PFA for 15 min at room temperature, followed by three PBS washes for 5 min each. Skulls were then stained in 2ml LacZ staining solution (Kferri/Kferro 5mM, MgCl₂ 2mM, X-gal 1mg/ml, NP-40 0.02%, Sodium Deoxycholate 0.01%) at 37°C for 8 h. Stained skulls were washed 3 times with PBS and then post-fixed in 4% PFA overnight at 4°C. Skulls were decalcified and embedded in paraffin; 6µM sections (described below) were counterstained with Neutral Red. Primary calvarial osteoblasts were digested from frontal and nasal bones (described below) and grown on glass coverslips overnight. Cells were fixed in 4% PFA with 0.4% glutaraldehyde at room temperature for 5 min and washed 3 times with PBS. Cells were stained in 1ml LacZ staining solution for 5–8 h, then washed 3 times with and treated with ProLong® Gold Antifade Reagent with DAPI (Life Technologies, P36931). LacZ positive cell number and bone perimeter were quantified with the multipoint tool and segmented line measurement function of Image J software. Expression of LacZ in live cells was examined using fluorescein di-β-D-galactopyranoside (FluoReporter LacZ Flow Cytometry Kit; Molecular Probes) staining according to the manufacturer’s instructions. Cells were analyzed on a FACSCanto (BD Biosciences) instrument.