Journal: PLoS Pathogens

Article Title: A Gammaherpesvirus Bcl-2 Ortholog Blocks B Cell Receptor-Mediated Apoptosis and Promotes the Survival of Developing B Cells In Vivo

doi: 10.1371/journal.ppat.1003916

Figure Lengend Snippet: WEHI immature B cell lines and control A20 mature B cells were plated in duplicate and induced to undergo apoptosis by exposure to anti-IgM (20 µg/ml). Non-induced counterparts for each cell line were included as negative controls. After 16 hours, whole cell lysates were prepared for Western blots or cells were collected for Annexin V staining. Western blots were performed to detect ( A ) the cleaved forms of caspase-9 (37 kDa) and caspase-6 (22 kDa), or ( B ) pro-caspase-3 (35 kDa) and cleaved caspase-3 (17 kDa). Whole cell lysates were collected and subjected to immunoblotting to detect caspases, as well as loading control actin (43 kDa). Densitometry plots indicate the relative volume (arbitrary units) of cleaved caspases after normalization to appropriate actin controls. Data shown is representative of 3 individual experiments.

Article Snippet: Bone marrow cells were stained with rat anti-mouse: CD19-allophycocyanin FITC (clone 1D3; BD Biosciences), AA4-APC (clone AA4.1; eBioscience), and IgM-PE-Cy7 (clone R6-60.2 BD Biosciences).

Techniques: Western Blot, Staining

Journal: PLoS Pathogens

Article Title: A Gammaherpesvirus Bcl-2 Ortholog Blocks B Cell Receptor-Mediated Apoptosis and Promotes the Survival of Developing B Cells In Vivo

doi: 10.1371/journal.ppat.1003916

Figure Lengend Snippet: Flow cytometric analyses were performed to determine the percent of apoptotic cells induced by anti-IgM or positive control actinomycin D (ActD, 40 nM) treatment. Cell samples were double-stained with Annexin V FITC and Sytox Blue, a nucleic acid stain that penetrates cells with compromised plasma membranes. Non-viable cells (Sytox Blue + ), viable cells (Annexin V − Sytox Blue − ), and apoptotic cells (Annexin V + Sytox Blue − ) were detected for all samples as shown. ( A ) Histograms from WEHI.EV samples presented for reference. ( B ) Bar graphs depict the percent of apoptotic cells after treatment with anti-IgM or positive control actinomycin D versus untreated controls. Data presented are the mean ± SD of two experiments with two samples per experiment. *P<0.05.

Article Snippet: Bone marrow cells were stained with rat anti-mouse: CD19-allophycocyanin FITC (clone 1D3; BD Biosciences), AA4-APC (clone AA4.1; eBioscience), and IgM-PE-Cy7 (clone R6-60.2 BD Biosciences).

Techniques: Positive Control, Staining

Journal: Protein & Cell

Article Title: Genetic approach to track neural cell fate decisions using human embryonic stem cells

doi: 10.1007/s13238-013-0007-y

Figure Lengend Snippet: NSC markers map to the mCherry Hi Population. Nestin:mCherry NPCs at P0 were stained with isotype control antibodies or FITC labeled anti-CD44, PE-Cy7 labeled anti-CD24, Alexa-647 anti-CD271 or APC labeled anti-CD184 antibodies. huES9 ES cells were used as an mCherry negative control. (A) CD184 + cells were gated on CD44 - CD24 + , while CD184 - were gated on CD24 - and both populations were shown relative to the CD184 and mCherry distributions. (B) CD271 - CD24 - and CD271 - CD24 + populations were shown with respect to CD44 and mCherry distributions

Article Snippet: For cell surface staining, cells were incubated with CD184-APC (560936; BD Pharmingen), CD24-PE-Cy7 (561646; BD Pharmingen), CD44-PE (51-9007231; BD Pharmingen) and/or CD271-Alexa fluor 647 or BV421 (560877 or 562562; BD Pharmingen) antibodies in PBS containing 1% bovine serum albumen (BSA) according to manufacturer’s protocol.

Techniques: Staining, Labeling, Negative Control

Journal: Protein & Cell

Article Title: Genetic approach to track neural cell fate decisions using human embryonic stem cells

doi: 10.1007/s13238-013-0007-y

Figure Lengend Snippet: mCherry Hi -based sorting strategy significantly enriches NSCs. (A) Nestin:mCherry NPCs at P0 were stained with isotype control antibodies or FITC labeled anti-CD44, PE-Cy7 labeled anti-CD24, Alexa-647 anti-CD271 or APC labeled anti-CD184 antibodies. mCherry Hi cells were gated on CD271 - CD44 - or CD44 - CD184 + and shown with respect to CD44 and CD24 distributions. (B) Nestin:mCherry NPCs at P0 were either unstained (Control) or stained with FITC labeled anti-CD44, BV421 labeled anti-CD271 and APC labeled anti-CD184 antibodies. The CD184 + CD44 - cells (all gating based on isotype control staining) were shown with respect to mCherry and CD271 distributions. The mCherry Hi CD271 - population was sorted for gene expression analyses. (C) mCherry Lo (Lo), mCherry Hi (Hi), mCherry Hi CD184 + CD271 - CD44 - (Hi +) populations were sorted from P0 Nestin:mCherry NPCs and real time PCR analysis was performed for Sox1, Sox2 and Pax6 expression relative to undifferentiated Nestin:mCherry huES9 hESCs. Results shown are from two independent experiments. * P < 0.05 and ** P < 0.01 (two-tailed T -test relative to the mCherry Lo condition)

Article Snippet: For cell surface staining, cells were incubated with CD184-APC (560936; BD Pharmingen), CD24-PE-Cy7 (561646; BD Pharmingen), CD44-PE (51-9007231; BD Pharmingen) and/or CD271-Alexa fluor 647 or BV421 (560877 or 562562; BD Pharmingen) antibodies in PBS containing 1% bovine serum albumen (BSA) according to manufacturer’s protocol.

Techniques: Staining, Labeling, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate, in acute myeloid leukemia

doi: 10.1158/1078-0432.CCR-16-1904

Figure Lengend Snippet: (A) Percentages of CD123+ and CD131+ fractions within CD45dim blast gate on primary AML samples are shown. (B) Samples were treated with SL-101 at indicated doses for 48 hours. Normalized viable cell counts (left) were determined in a cohort of primary AML samples. (C) Gating scheme for the LSC population (CD45dimCD34+CD38−CD123+). Specific apoptosis was calculated based on percentage of Annexin-V+ cells using the following formula: percentage of specific apoptosis = 100 × (% apoptosis of treated cells − % apoptosis of control cells)/(100 − % apoptosis of control cells). Percentage of growth inhibition was calculated based on Annexin-V−/DAPI− viable cells using the following formula: 100 – 100 × % viable cells of treated cells/% of viable cells of control cells in both CD45dim blasts and LSC-enriched blasts. (D) Colony-forming cell assay was performed in primary AML samples and normal bone marrow (NBM) cells. Data were normalized to those from untreated control groups and represent the mean values of triplicate assays. The absolute colony numbers were shown in Supplementary Figure 1.

Article Snippet: MNCs isolated from primary AML specimens were stained with a cocktail of antibodies comprising CD34-FITC (Cat. 555821), CD38-PE-Cy7 (Cat. 335790), CD45-APC-Cy7 (Cat. 557833), CD123-PerCP-Cy5.5 (Cat. 558714; all from BD Biosciences), and Annexin-V–APC for 30 minutes at room temperature in dark.

Techniques: Inhibition

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate, in acute myeloid leukemia

doi: 10.1158/1078-0432.CCR-16-1904

Figure Lengend Snippet: The primary AML patient sample 11 (4030094) was left untreated or pre-treated with SL-101 (1.0 μg/mL) overnight prior to transplantation via intravenous injection into NSG mice (1×106 viable cells for each mouse). (A) Engraftment of human AML cells was measured by flow cytometry in blood by using anti-human CD45 antibody at week 5 after injection (left). CD123+ cells were gated on the human CD45+ fraction (right). (B) At week 6, all mice were sacrificed and engraftment of human AML was measured in both bone marrow (BM) and spleen. (C) Representative spleens from untreated (U) and SL-101-treated (S) mice are shown. (D) The gating scheme is shown for the CD45dimCD34+CD38−CD123+ LSC-enriched population in untreated and SL-101–treated mice. (E) Percentages of LSCe in human CD45+ cells were determined in both bone marrow and spleen by flow cytometry. AML11 cells (0.6×106) were injected into NSGS) mice that were treated intravenously with PBS or SL-101 at 0.1 mg/kg every other day for 6 doses, after confirming human CD45 engraftment. (F) Leukemia burden was measured after treatment (G) Body weight was monitored over treatment.

Article Snippet: MNCs isolated from primary AML specimens were stained with a cocktail of antibodies comprising CD34-FITC (Cat. 555821), CD38-PE-Cy7 (Cat. 335790), CD45-APC-Cy7 (Cat. 557833), CD123-PerCP-Cy5.5 (Cat. 558714; all from BD Biosciences), and Annexin-V–APC for 30 minutes at room temperature in dark.

Techniques: Transplantation Assay, Injection, Flow Cytometry