Journal: Springer Protocols Handbooks

Article Title: Protocols for Neural Cell Culture

doi: 10.1007/978-1-60761-292-6

Figure Lengend Snippet: Fig. 5.4. Human fetal CNS cells. Representative image of oligodendrocyte progenitor cells labeled with A2B5; they are also immunopositive for PDGFR and are typically bipolar. Neurons are labeled with TubIII (green) and grow on top of GFAP-positive astro- cytes (red); neurons are also immunopositive for MAP-2 and neurofilaments. Astrocytes are labeled with GFAP. Microglia are demonstrated as a bright-field image, and are typ- ically immunopositive for CD68, MHC-II, CD11c, and CD45. Scale bars, 50 m.

Article Snippet: A2B5 hybridoma cells (ATCC, CRL-1520, glycolipid anti- gen clone 105, IgM) 4.

Techniques: Labeling

Journal: Springer Protocols Handbooks

Article Title: Protocols for Neural Cell Culture

doi: 10.1007/978-1-60761-292-6

Figure Lengend Snippet: Fig. 5.5. Human adult CNS cells. Representative image of mature oligodendrocytes labeled with MAG; these cells are also immunopositive for MBP, MOG, NogoA, and GalC. Microglia are represented as a bright-field image and are immunopositive for CD68, MHC-II, CD11c, and CD45. Oligodendrocyte progenitor cells are labeled with A2B5 and are also immunopositive for another progenitor marker, PDGFR; a subset of these cells is immunopositive for GalC, MAG, and NogoA. Scale bars, 50 m.

Article Snippet: A2B5 hybridoma cells (ATCC, CRL-1520, glycolipid anti- gen clone 105, IgM) 4.

Techniques: Labeling, Marker

Journal: PLoS ONE

Article Title: Pluripotent Transcription Factors Possess Distinct Roles in Normal versus Transformed Human Stem Cells

doi: 10.1371/journal.pone.0008065

Figure Lengend Snippet: (A–D) Representative images of colonies generated from GFP + SSEA3 + cells 11 days post-sort from control (A and B) and Oct4 knockdown (C and D) normal hPSCs. Scale bar = 100 µm, n = 3. Note the compact appearance and well-defined border of the typical hPSC colony in A and B. In contrast, the cells within the more differentiated colony in C and D exhibit a higher cytoplasm to nuclear ratio, are less compact, and do not form a well-defined colony border. (E) qPCR of fold changes in Oct4, Nanog, Sox2, c-Myc, Dpp4a, and Tbx3 transcripts in Oct4 knockdown hPSCs relative to hPSC eGFP controls. Bar graphs represent mean values ± SEM, n = 3, ***, p<0.001. (F) Clonogenic self-renewal of SSEA3 + cells isolated from control (39.3±4.3×10 4 ) and Oct4 knockdown (14±2.3×10 4 ) hPSCs. 1×10 4 GFP + SSEA3 + cells were isolated from hPSCs 2 days after transduction with eGFP control and Oct4 knockdown lentiviral vectors and seeded on ihdFs. GFP + colonies were scored 9 days after seeding. Bar graphs represent mean values ± SEM, n = 3. **, p<0.01. (G–I) Frequency of GFP + SSEA3 + (G) GFP+A2B5+ (H) and GFP+AnnexinV+ (I) cells for three passages of culture derived from sorted GFP + SSEA3 + fractions of control and Oct4 knockdown hPSCs. N = 3 for each. Line graphs represent mean values ± SEM. * p<0.05, ** p<0.01. (J–M) Representative images of colonies generated from sorted GFP + cells 6 days post-sort from control (J–K) and Oct4 knockdown (L–M) t-hPSCs. Scale bar = 100 µm, n = 5. Arrow denotes the typical appearance of tightly packed undifferentiated colonies. (N) qPCR of fold changes in Oct4, Nanog, Sox2, c-Myc, Dpp4a, and Tbx3 transcripts in Oct4 knockdown t-hPSCs relative to t-hPSC eGFP control cells. Bar graphs represent mean values ± SEM, n = 3, **, p<0.01, ***, p<0.001. (O) Clonogencity of GFP + cells isolated from control and Oct4 knockdown t-hPSCs. 1×10 4 GFP + cells were sorted from t-hPSCs 4 days after transduction with eGFP control and Oct4 knockdown lentiviral vectors and seeded on ihdFs. GFP + Colonies were scored 6 days after. Bar graphs represent mean values ± SEM, n = 3. (P–R) Frequency of GFP + SSEA3 + (P) GFP+A2B5+ (Q) and GFP+AnnexinV+ (R) cells for three passages of culture derived from sorted GFP + fractions of control and Oct4 knockdown t-hPSCs. P, n = 9. Q, n = 5, R, n = 3. Line graphs represent mean values ± SEM.

Article Snippet: A2B5 was detected with antibodies A2B5 (R&D systems) and Alexa fluor 647 goat anti mouse IgM (Invitrogen).

Techniques: Generated, Control, Knockdown, Isolation, Transduction, Derivative Assay

Journal: PLoS ONE

Article Title: Pluripotent Transcription Factors Possess Distinct Roles in Normal versus Transformed Human Stem Cells

doi: 10.1371/journal.pone.0008065

Figure Lengend Snippet: (A–B) Representative images of normal hPSC bulk culture one week after transduction with a lentivector carrying an shRNA sequence targeting Nanog. Scale bar = 100 µm, n = 5. A: Phase contrast. B: GFP. Note the less compact colony morphology and the differentiated interior. Arrow denotes differentiated cells in the hPSC colony centre following Nanog knockdown. (C) qPCR demonstrating fold changes in Oct4, Nanog, Sox2, c-Myc, Dpp4a, and Tbx3 transcripts in GFP + SSEA3 + cells isolated from Nanog knockdown hPSCs relative to the control, respectively. Bar graphs represent mean values ± SEM, n = 3. **, p<0.01, ***, p<0.001. (D) Clonogenic self-renewal of SSEA3 + cells isolated from control and Nanog knockdown hPSCs. 1×10 4 GFP + SSEA3 + cells were isolated from hPSCs 2 days after transduction with eGFP control and Nanog knockdown lentiviral vectors and seeded on ihdFs. GFP + colonies were scored 9 days after seeding. Bar graphs represent mean values ± SEM, n = 3. **, p<0.01. (E–G) Frequency of GFP + SSEA3 + (E) GFP+A2B5+ (F) and GFP+AnnexinV+ (G) cells for three passages of culture derived from sorted GFP + SSEA3 + fractions of control and Nanog knockdown hPSCs. Lines represent mean values ± SEM, n = 3. * p<0.05, ** p<0.01. (H–I) Representative images of t-hPSC bulk culture one week after transduction with a lentivector carrying an shRNA sequence targeting Nanog. Scale bar = 100 µm, n = 5. H: Phase contrast. I: GFP. Arrow denotes t-hPSCs undergoing apoptosis following Nanog downregulation. (J) qPCR demonstrating fold changes in Oct4, Nanog, Sox2, c-Myc, Dpp4a, and Tbx3 transcript in GFP + cells isolated from Nanog knockdown t-hPSCs. Bar graphs represent mean values ± SEM, n = 3, *, p<0.05, **, p<0.01, ***, p<0.001. (K) Clonogenic self-renewal of GFP + cells isolated from control and Nanog knockdown t-hPSCs. 1×10 4 were sorted from t-hPSCs 4 days after transduction with eGFP control and Nanog knockdown lentiviral vectors and seeded on ihdFs. GFP + colonies were scored 6 days after seeding. Bar graphs represent mean values ± SEM, n = 3. **, p<0.01. (L–N) Frequency of GFP + SSEA3 + (L) GFP+A2B5+ (M) and GFP+AnnexinV+ (N) cells for three passages of control and Nanog knockdown t-hPSCs. Lines represent mean values ± SEM, n = 3. * p<0.05.** p<0.01.

Article Snippet: A2B5 was detected with antibodies A2B5 (R&D systems) and Alexa fluor 647 goat anti mouse IgM (Invitrogen).

Techniques: Transduction, shRNA, Sequencing, Knockdown, Isolation, Control, Derivative Assay

Journal: American Journal of Cancer Research

Article Title: Isolation and characterization of cancer stem cells derived from human glioblastoma

doi:

Figure Lengend Snippet: Differentiation of U-251MGSC1 cells in the presence of the condition medium from U-251MG cells. Immunofuluorescent cytochemistry of U-251MGSC1 cells against neural stem cell markers, A2B5 and Nestin (A) and neural cell markers, GFAP and NF-M (B) after exposed to 10% and 50% of CM for 2 weeks. 50% CM decreased the neural stem cell markers and enhance the neural cell markers suggesting the differentiation. Scale bar =53 µm.

Article Snippet: Anti-GFAP antibody (J0510, Santa Cruz Biotechnology, CA) (dilution 1:50), anti-Nestin antibody (N6413, Sigma Aldrich, MO) (dilution 1:100), anti-NF-M antibody (BML-NA1216, Enzo, NY) (dilution 1:50) and anti-A2B5 antibody (130-097-864, Miltenyi Biotec, CA) (dilution 1:50).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Regulatory B Cells Normalize CNS Myeloid Cell Content in a Mouse Model of Multiple Sclerosis and Promote Oligodendrogenesis and Remyelination

doi: 10.1523/JNEUROSCI.2840-19.2020

Figure Lengend Snippet: Oligodendroglial lineage after adoptive transfer with B cells or Bregs in EAE mice. Mice were killed starting 2 weeks after adoptive cell transfer, and spinal cords were collected for analysis by flow cytometry and Western blotting. Naive mice and untreated EAE mice were used as control. A, Gating strategy: single cells in side and forward scatter, cells, live cells followed by CD45–/low gate. B, Expression of oligodendroglial markers 2 (and 3.5 weeks for Bregs** treated animals) after adoptive transfer with Bregs or B cells compared with WT and EAE-untreated mice: A2B5, CD140, GalC, and O1 in WT mice and EAE C57BL/6 treated either with B cells or Bregs. C, Statistical analysis of the oligodendroglial marker expression in the spinal cords and brains 2 weeks after adoptive transfer. D, Oligodendroglial phenotyping from spinal cords in WT, Bregs, and B-cell-treated mice: A2B5+CD140+, early OPCs, and O1+GALC+ mature oligodendrocytes. E, Western blotting analysis on the expression of MOG, MBP, and paired-related homeobox protein 1 (PRXX1) by spinal cord oligodendrocytes from EAE C57BL/6 mice untreated, treated with Bregs or B cells, and WT mice 2 weeks after adoptive transfer, load control GADPH. Data are representative of three independent experiments in vivo with n ≥ 6. Data are mean ± SEM. *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001; compared with the corresponding control (Student's t test).

Article Snippet: For oligodendrocyte analysis, the following markers were used as per the manufacturer's recommendation: R&D Systems anti-A2B5 AlexaFluor-647 (clone #105), R&D Systems anti-oligodendrocyte marker O1 AlexaFluor-405-conjugated antibody, Sigma Millipore anti-galactocerebroside FITC antibody (clone mGalC), and Invitrogen anti-CD140 (PDGFRA) FITC (clone APA5).

Techniques: Adoptive Transfer Assay, Flow Cytometry, Western Blot, Expressing, Marker, In Vivo