Flow cytometry was performed to quantify the expression of cell-specific markers in both freshly isolated and pre-induced hPBMCs. Both direct and indirect immunolabeling methods were used. Antibodies used for direct labeling (FITC- or PerCP-conjugated) were specific for human CD3, CD16, CD19, or CD45 (Invitrogen, USA), and each staining trail was compared to a specific isotype control. Primary antibodies used for indirect labeling were specific for human nestin (Millipore, USA), vimentin, MAP2, GFAP, synapsin, rhodopsin (all from Abcam, England), or β-tubulin III (Sigma-Aldrich, USA). The secondary antibodies were FITC- conjugated goat anti-rabbit (Cell Signaling Technology, USA) or PE-conjugated goat anti-rabbit (SouthernBiotech, USA). Fix and Cell Permeabilization reagents (Invitrogen, USA) were used for labeling intracellular antigens. Cell suspensions were stained and counted by flow cytometry according to the manufacturer’s directions. Cell suspensions treated with secondary antibodies only were measured as the isotype controls for indirect labeling. After staining, cell suspensions were tested immediately by flow cytometry (Becton, Dickinson Company, USA) using FCS Express V3 software for data analysis. The positive value of the isotype controls ranged from 0% to 1%.
Human nestin
Manufactured by Merck Group
Sourced in United States
Human Nestin is a protein that is primarily expressed in stem and progenitor cells. It is a type VI intermediate filament protein that plays a role in the maintenance and differentiation of these cells. Human Nestin is commonly used as a marker for identifying and studying stem and progenitor cell populations in various tissues and developmental processes.
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Lab products found in correlation
Ctip2, by Abcam (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Rhodopsin, by Abcam (1 mentions)
β tubulin 3, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
Alexa 555 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Islet1, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Musashi, by Abcam (1 mentions)
Tra 1 81, by Merck Group (1 mentions)
Vglut1, by Synaptic Systems (1 mentions)
6 protocols using human nestin
1
Flow Cytometry Characterization of hPBMCs
2
NSC Fate Determination in Mouse Brain
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For determination of the fate of NSCs in non-tumor-bearing mouse brain, NSG mice were injected with 5 × 105 NSCs (n = 6) intracranially and were euthanized at 1, 4, and 12 weeks after injection, after which their brains were harvested. The brains were then isolated, fixed, and stained (every tenth section) with H&E (American Master Tech Modified Mayer's H&E), Ki-67 (Dako), TUNEL (Chemicon), and human NESTIN (Millipore). Quantification of percentile Ki-67-positive cells was performed using previously stained sections relative to the dose on LM-NSC008 cells injected.
3
Immunofluorescence Characterization of NPCs
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To analyze the marker expression of NPCs or neurosphere-derived neural cells in microfluidic devices, the devices were fixed in 4% paraformaldehyde for 15 min and washed three times in Ca2+-, Mg2+-containing phosphate-buffered saline (PBS; Welgene, Korea). Primary antibodies used in this study were human Nestin (1:200, Chemicon) and type III β-tubulin (Tuj1) (1:500, Chemicon). Secondary antibodies used were goat anti-mouse IgG-conjugated Alexa-555 (1:200, Molecular Probes, USA) and goat anti-rabbit IgG-conjugated Alexa-488 (1:200, Molecular Probes). Following the secondary antibody reaction, nuclei of cells were counterstained with DAPI (1:1000, Roche). Subsequently, they were mounted in Vectashield™ (Vector Laboratories, USA). Stained NPCs and neurons were examined and photographed using a confocal laser scanning microscope imaging system (LSM510, Carl Zeiss, Inc., USA).
4
Immunofluorescence Analysis of Neural Markers
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Cells were briefly fixed in 4% paraformaldehyde for 10 min and then permeabilized with 0.5% Triton-X100 in PBS. Cells were then blocked in 0.5% Triton-X100 with 5% donkey serum for 1 hr before incubation with primary antibody overnight at 4°C. After three washes in PBS, cells were incubated with secondary antibodies (Jackson ImmunoResearch) for 1 hr at room temperature. Fluorescent signals were detected using a Zeiss LSM 710 Laser Scanning Confocal Microscope and images were processed with Photoshop CS3 (Adobe Systems). Primary antibodies used in this study were human Nestin (1:100, Chemicon); Tuj-1 (1:500, Covance); Map2 (1:100, Sigma); GFP (1:200, Molecular Probes-Invitrogen) Synapsin1 (1:200, Calbiochem), GABA (1:200, Sigma) and CTIP2 (1:200, Abcam).
5
Immunocytochemical Analysis of Differentiated Cells
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After 14 days of differentiation, the cells were fixed in acetone at −20°C for 2 min. The fixed cells were blocked with 5% sheep serum for 10 min at room temperature and then incubated for 90 min at room temperature with human Nestin (1:300; Chemi-Con, USA), Islet-1 (cat# SC30200, Santa Cruz, USA), and AChE (cat# 3739, Abcam, USA). Subsequently, the cells were washed three times with 0.1% PBS/BSA and incubated with Fluorescein Isothiocyanate (FITC)-labeled sheep anti-rabbit IgG or sheep anti-mouse IgG at room temperature for 45 min in the dark. The nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride at 1 μg/mL for 5 min. After washing, the coverslips were mounted in 70% PBS-glycerol and examined under a fluorescence microscope (Olympus, Tokyo, Japan).
6
Immunofluorescence Staining of Stem Cells
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The cells were fixed with PBS containing 4% paraformaldehyde for 10 minutes and then incubated at room temperature for 1 hour in a blocking solution containing 5% donkey serum and 0.1% Triton X-100. The primary antibodies were incubated overnight at 4°C, followed by incubation with secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature. Images were captured with a Zeiss microscope. The primary antibodies used included the following: Tra-1-81 (1:100, Chemicon); Nanog and Lin28 (1:500, R&D Systems); Sox2 (1:250; Chemicon); human Nestin (1:100, Chemicon); Tuj1 (1:500, Covance); MAP2 (1:100; Sigma); VGLUT1 (1:200, Synaptic Systems); GABA (1:100, Sigma); Musashi (1:200, Abcam); Ctip2 (1:200, Abcam); and Tbr1 (1:200, Abcam).
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