Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Summary of Primary Antibodies Used in These Studies

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Generated

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Human neuronal precursors were mitotic and E-NCAM+. First-passage cells were acutely dissociated, plated, and processed for immunocytochemistry 48 hr later. A: First-passage HNPs displayed IR for E-NCAM (A, red) and β-III tubulin (A″, green). DAPI staining of nuclei (A′, blue) shows that only a subset of the cells expresses neuronal markers. β-III Tubulin+ and E-NCAM+ cells (B,C, green) incorporated BrdU (B,C, red), indicating that they were mitotic (arrowheads indicate double-labeled cells). β-III Tubulin+ cells (D, red) coexpressed another neuronal-specific protein, MAP2 (D′, green). β-III Tubulin+ cells (F, green, and G, red) did not coexpress the glial markers A2B5 (F, red) or GFAP (G, green). Staining was performed on two or three dishes of cells from four different isolations. E shows human NRP antigenic profiles.

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Immunocytochemistry, Staining, Labeling

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Immunostaining Profiles (%) of Human Fetal Brain Culture *

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Immunostaining

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Immunostaining Profiles (%) of Differentiated E-NCAM + HNPs *

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Immunostaining

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: HNPs do not coexpress nestin. Acutely dissociated human neural cells were grown for 5 days in culture, pulsed with BrdU for 16 hr (E,F), fixed, and processed for immunocytochemistry to detect the expression of nestin (A,B,D,F, red, and C,E, green) and GFAP (B, green), E-NCAM (C, red), β-III tubulin (D,F green), and BrdU (E, red, and F, blue). DAPI staining (A, blue) was used to identify all cells. Large numbers of nestin+ cells were present in culture and made up about 70% of the total cell population (A). About 15% of the nestin+ cells coexpressed GFAP IR (B, arrowheads), although a few GFAP+ cells did not express nestin (B, arrows). Five percent of the E-NCAM+ cells were nestin+ (C, arrowheads), but none of the β-III tubulin+ cells was nestin+ (D). BrdU labeling identified dividing cells (E,F) and showed that BrdU incorporation was seen in both nestin+ (E,F, arrowhead) and nestin− (E,F, arrow) cells. Triple labeling showed that some of the nestin−, BrdU+ cells (F, arrowheads) were β-III tubulin+ (green). Only 5% of the β-III tubulin+ cells incorporated BrdU, and postmitotic BrdU−, nestin−, β-III tubulin+ cells were also seen (F, arrow, green). Staining was performed on two or three dishes of cells from four different isolations.

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Immunocytochemistry, Expressing, Staining, Labeling, BrdU Incorporation Assay

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Differentiating HNPs expressed synaptophysin and neurotransmitter-synthesizing enzymes. Cells were maintained under differentiating conditions for 14 DIC and processed for immunocyto-chemistry for E-NCAM (A), synaptophysin (A′), glutamate (B), glycine (C), TH (D), and ChAT (E). A single cell exhibiting synaptophysin IR is shown (A′), and expression colocalizes with that of E-NCAM (A). Subsets of cells express IR for glutamate (B, arrowhead), glycine (C, arrowhead), TH (D, arrowhead), and ChAT (E, arrowhead), whereas other cells are negative (B–E, arrows). Staining was performed on two or three dishes of cells from two different isolations.

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Expressing, Staining

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: E-NCAM+ HNPs immunoidentified for patch-clamp and fura-2 recordings. Shown are mixed human cell cultures from undifferentiated conditions (A) or following 14 days under differentiating conditions (B), visualized using Hoffman optics. The cells were live stained for E-NCAM (red in A′, B′). A composite Hoffman-fluorescence image (A″, B″) shows the red E-NCAM+ HNPs from which whole-cell currents were recorded (arrows). Identical methods were used to identify E-NCAM+ cells used for Ca2+ imaging experiments.

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Patch Clamp, Staining, Fluorescence, Imaging

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Differentiating HNPs expressed voltage-gated Na+ and K+ channels and fired action potentials. A: Whole-cell voltage-clamp recordings were made from E-NCAM+ HNPs in acutely passaged conditions (A1,A2) or following differentiation (A3). Cells were held at −100 mV and stepped to test voltages between −80 and 80 mV in 10 mV increments. Both of the acute HNPs expressed outward K+ currents (A1,A2), but only one exhibited a small inward Na+ current (A2). A differentiated HNP expressed both outward K+ and inward Na+ currents (A3). B1–3: Peak outward K+ currents (triangles) and peak inward Na+ currents (circles) were plotted against the command voltage for the cells represented in A. C: HNPs under acutely passaged conditions failed to fire action potentials (C1,C2); in contrast, a differentiated HNP fired an action potential when stimulated with depolarizing current injections (C3). Inset in C3 shows the action potential overshoot on a longer time scale.

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques:

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Differentiating HNPs expressed functional neurotransmitter receptors. A,B: Ratiometric imaging of acute and differentiating E-NCAM+ HNP cells. Ratio of fura-2 emission (I340/I380, left y axis) with approximate Ca2+ concentrations ([Ca2+]i, right y axis). Arrow-heads and labels show application of neurotransmitters. All substances were applied at 500 μM, except for K50, which was 50 mM K+ HR. A: An acutely passaged HNP responded only to acetylcholine (ACh), with small magnitude. B: A differentiated neuron responded to GABA, glutamate (E), glycine (G), elevated K+ (K50), and ACh. C: Fraction of cells responding to neurotransmitters is shown for acutely passaged cells (open bars, n = 17) and differentiated cells [shaded bars, n = 70, except for norepinephrine (NE), for which n = 43]. Acute HNPs did not respond to GABA, glycine, dopamine (DA), NE, or ascorbic acid (AA) control. Differentiated neurons responded to all substances with higher frequencies, but only ACh and NE were significant (asterisks). D: The distribution of response amplitudes is shown by the box plot of acute (left box, open circles) and differentiating cells (Diff; right box, gray circles). Squares represent distribution mean, box represents the 75th percentiles, error bars represent the 95th percentiles, and circles plot the individual response amplitudes. Experiments were performed on a total of six dishes of cells from three different isolations.

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Functional Assay, Imaging, Control

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Subset of E-NCAM- and β-III tubulin-immunoreactive HNPs coexpresses α-tubulin promoter-driven GFP. Tα1:GFP constructs were transfected into day 2 cultures of fetal neural cells. GFP expression (A,A′, A″, B,C,D, green) was seen in cells with neuronal morphology as early as 24 hr after transfection. Plates were fixed and processed for E-NCAM IR (B, inset, B′, red), β-III tubulin (C, red), and GFAP (D, red). The inset shows lack of colocalization for Tα1:GFP (green) and E-NCAM (red). We show one Tα1:GFP+ cell that was E-NCAM+ (B,B′, arrowhead) and one that was β;-III tubulin+ (C, arrowhead). None of the Tα1:GFP-expressing cells was GFAP+ (arrows in C and D show lack of colocalization). These experiments were performed on two or three dishes of cells from three different isolations.

Article Snippet: E-NCAM , IgM , Hybridoma , Monoclonal , 1:1 , Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA.

Techniques: Construct, Transfection, Expressing