Purity of freshly isolated CNS capillary suspensions. Freshly isolated CNS capillary suspensions were enzymatically disrupted as detailed in Methods (DoreDuffy, 2003). After disruption, singlecell suspensions were stained using immunocytochemical techniques as well as the antigenic properties of cell populations analyzed by flow cytometry. CNS capillary digests contained cells that bound FITCGSA lectin (ECs) and stained positively for your expression of vWF (data not shown during this figure). Preparations also contained nestinpositive cells and NG2positive cells. Capillary preparations wouldn't contain O4+ cell populations, cells that stained positive for expression of neurofilament (NF), or cells that expressed GFAP. Cultured FACS sorted GSA cells expressed SMA (data not shown in this figure).
Figure 2.
Characterization of nestin/NG2positive cells in CNS capillaries. Freshly isolated CNS capillaries were dual stained for expression of vWF(green)/nestin(red) (A); NG2(red)/nestin(green) (B); and NG2(red)/vWF(green) (C). Nuclei were stained with DAPI. Freshly isolated CNS capillaries were Cheap Louboutin shoes enzymatically digested as detailed in Methods and cell suspensions stained for expression of nestin along with the potential to bind FITCconjugated GSA lectin and analyzed by flow cytometry (D). Enzymatically disrupted cell suspensions red bottoms were also would once culture primary pericytes (DoreDuffy, 2003). Primary pericytes were dual stained for expression of SMA(red)/nestin(green) (E) and SMA(red)/NG2(green) (F).
Figure 3.
Expression of nestin and NG2 transcripts in cultured CNS primary pericytes. Freshly isolated CNS capillaries were enzymatically digested and pericytes subcultured as detailed in Methods. Primary pericytes were cultured in DMEM plus FCS and N2bFGF medium. Total RNA Cheap Christian Louboutin shoes was isolated from capillaries pericytes and from pericyte derived spheres. Reverse transcriptase polymerase incidents analysis revealed that capillaries (lane 1), pericytes (lane 2), and spheres (lane 3) expressed mRNA transcripts for nestin (A, top left) and NG2 (B, bottom left). Primary pericytes were also cultured for varying lengths of time as many as 21 days in DMEM plus serum. Nestin and NG2 transcripts were analyzed by quantitative realtime PCR (right panels). Results indicated that pericyte christian louboutin outlet expression of nestin decreased with time in culture (top right) while NG2 mRNA expression remained relatively stable (bottom right) over the culture period.
Figure 4.
Primary CNS pericytes are responsive to bFGF. Primary CNS pericytes were cultured in medium with N2 supplement and bFGF for variable amounts of time as detailed in Methods. Almost all cells remained in suspension although some adhered to the culture dish. Both adhered and floating cells replicated forming spheres that increased in size (A Cells at 2 to 4 days in culture are shown in (A), Seven days in (B) and a couple to 3weekold spheres in (C). Threeweekold spheres were nestin+ (D) and labeled with BRDU (E). Nuclei were stained with DAPI both in (E) and (D).
Figure 5.
Characterization of differentiated pericytederived spheres. Two to threeweekold pericytederived spheres were known as immunocytochemistry (top panels) and flow cytometry (bottom panels). Pericytederived spheres contained cells that stained positively for dual expression of nestin/GFAP (top row, top panels); nestin/04 (middle row, top panels) and nestin/NF (bottom row, top panels). Pericytederived spheres were disrupted, stained and analyzed by flow cytometry (bottom section, right panel). Primary rat astrocyte preparations were similarly analyzed as controls(bottom section, left panel). FACS analysis revealed that spheres showed evidence differentiation on the neural lineage. Pericyte derived spheres contained cells that expressed GFAP, NF and O4+. Astrocyte control cell suspensions expressed GFAP, a % of 04 positive cells no cells expressing NF.
Figure 6.
Antigenic properties of cells cultured from pericyte derived spheres. CNS primary pericytederived spheres (A three-week period old) were disrupted and cultured for to 7-day period in DMEM supplemented with 20% serum. Cells were dual stained for expression of indicated markers using immunocytochemistry. Nuclei were stained with DAPI. Differentiated spheres christian louboutin shoes yielded cells that exhibited expression of: SMA(red)/nestin(green); SMA(red)/NF(green), NF(green); SMA(red)/GFAP(green); and SMA(red)/04(green) (top left to right). Spheres also generated SMA(red)/NG2(green) positive cells (bottom left) who were also nestin positive (not shown). Interesting is the fact spheres generated cells that exhibited dual expression of GFAP(green)/04(red), 04(green)/NF(red) and GFAP(green)/NF(red) (bottom panel left to right). Spheres also generated cells that expressed only NF (welcomed in the top end panel second from left)seen by 4 days in culture.
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