Journal: Frontiers in Immunology

Article Title: Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies

doi: 10.3389/fimmu.2022.847008

Figure Lengend Snippet: NK cells rely on endogenous IL15 production for in vivo persistence in NSG mice. (A) Schematic representation of the vector maps of the IL15 constructs used for NK cell transduction. (B) Primary human NK cell preparations consisting of 50% EGFP/IL15 or EGFP/IL15-IL15R and 50% untransduced NK cells (or 100% untransduced cells as control group) were injected into busulfan-preconditioned mice (3 animals per group). On days 5, 10, 20, and 27, NK cell persistence in the bloodstream was analyzed by mouse CD18, human CD45, and CD56 staining as well as EGFP expression. (C) Example of gating strategy showing representative data from day 10. Values indicate mean ± SEM of all three animals. (D) NK cell persistence in the blood including ratios of transduced to untransduced NK cells. Data are represented as mean ± SEM. Significances of p<0.05 are indicated by an asterisk (*).

Article Snippet: NK cell purity and phenotype were determined by flow cytometry (MACSQuant ® Analyzer 10, Miltenyi Biotec, Bergisch Gladbach, Germany) on days 0, 7, and 14, using fluorochrome-conjugated antibodies against CD3, CD33, CD56, and CD94 (all REAfinityTM clones from Miltenyi Biotec).

Techniques: In Vivo, Plasmid Preparation, Construct, Transduction, Control, Injection, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies

doi: 10.3389/fimmu.2022.847008

Figure Lengend Snippet: CAR NK cells rely on endogenous IL15 production for leukemia control in an ALL xenograft model. (A) Schematic representation of vector maps of CAR constructs used for NK cell transduction. (B) Primary human NK cell preparations consisting of 50% BFP/CD19 CAR, IL15/CD19 CAR, or IL15-IL15R/CD19 CAR and 50% non-transduced NK cells were injected 2 days after xenotransplantation of REH LucEG into busulfan-preconditioned NSG mice (6 animals per group, 7 animals as untreated control). (C) On days 7, 15, 22, and 27, leukemia progression was analyzed by luminescence imaging in a Caliper device. (D) Kaplan–Meier survival curves for the three treatment groups and the untreated control animals. (E) REH LucEG and (F) NK cell persistence in the bloodstream was analyzed by CD19, CD34, CD45, and CD56 staining as well as EGFP expression by flow cytometry. Data are represented as mean ± SEM. Significances of p<0.05 are indicated by an asterisk (*).

Article Snippet: NK cell purity and phenotype were determined by flow cytometry (MACSQuant ® Analyzer 10, Miltenyi Biotec, Bergisch Gladbach, Germany) on days 0, 7, and 14, using fluorochrome-conjugated antibodies against CD3, CD33, CD56, and CD94 (all REAfinityTM clones from Miltenyi Biotec).

Techniques: Control, Plasmid Preparation, Construct, Transduction, Injection, Imaging, Staining, Expressing, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models

doi: 10.1038/s44321-024-00055-9

Figure Lengend Snippet: ( A ) NSG-SGM3 mice were engrafted intravenously (i.v.) with 1 × 10 6 EBFP- and luciferase-expressing NALM6 tumor cells. Tumor load was monitored by in vivo bioluminescence imaging system (IVIS). In all, 1 × 10 7 stCAR T cells or an equal amount of T cells were injected i.v. on day 0 and mice were monitored for general health conditions until termination criteria were fulfilled. ( B ) Body temperature change was determined as difference to experimental start at day −10. Similarly, weight was normalized to the start weight. Average values for all three groups are shown. Red dotted lines indicate the thresholds for temperature changes rated as critical adverse event. ( C ) Individual dot plot presentation of human CD3 + T cells in blood of each mouse. ( D ) Bar diagrams show the frequencies of human T cells within viable cells (left) and absolute numbers per µL blood one day after stCAR T-cell or T-cell administration (right). ( E ) Human plasma cytokines one day after treatment. ( F ) Composition of murine cells in spleen. ( G ) Murine plasma cytokines determined on the respective termination days, which were one day after treatment for the stCAR T-cell group and 12 days after injection for the T-cell and PBS groups. Data information: Bar diagrams provide data points for each mouse with mean and standard deviation of the group. n = 4 for T-cell and stCAR T-cell groups, n = 2 for PBS group. Cytokine concentrations and body weights are the average values of two, body temperatures of three technical replicas. Statistics were assessed by unpaired t test and P values are indicated. .

Article Snippet: The following anti-human antibodies were used for FACS staining: CD45 (2D1, BV510, BioLegend or 5B1 VioBlue Miltenyi Biotec), CD3 (BW264/56, PerCP, Miltenyi Biotec; HIT3a, BV605, BD Bioscience), CD14 (REA599, APC, or Tuk, Percp both Miltenyi Biotec), CD4 (L200,PE, BD Bioscience, VIT-4, PerCp or VioBlue, Miltenyi Biotec), CD8 (RPA-T8, BV786, BD Bioscience; BW135/80, APC or PE Miltenyi Biotec).

Techniques: Luciferase, Expressing, In Vivo, Imaging, Injection, Clinical Proteomics, Standard Deviation

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Representation of the strategy used for transcriptomic analysis in time and space in the dorsal and lateral OB lineages. pCX-GFP plasmid was introduced into neural stem cells (NSCs) residing within the dorsal or lateral V-SVZ and GFP-positive cells were isolated by FACS at different time points after electroporation (Elpo). The mRNA content was analyzed by micro-array . ( B ) Quantification of Vax1 mRNA expression detected by micro-array analysis in dorsal (brown) and lateral (purple) progenies during neurogenesis. ( C–H ) In situ hybridization revealing Vax1 mRNA (in blue) combined with immuno-histochemistry using antibodies detecting (in brown) PAX6 ( C, C’, G ), ASCL1 ( D, D’ ), DLX2 ( E, E’, H ) or KI67 ( F, F’ ) proteins in the V-SVZ ( C–F ) or RMS ( G, H ) at postnatal day 3 ( P3 ). ( C’–F’ ) High magnification of cellular staining in the V-SVZ (area indicated by the yellow bracket in C-F). Arrows ( C’ ): examples of strong PAX6 only positive cell in the dorso-lateral SVZ; blue staining underneath labels cells from a distinct plane. Arrow heads ( E’, F’ ): double positive cells for DLX2 and KI67, respectively. High magnification of the RMS highlights the differential expression of Vax1 and Pax6 along the dorso-ventral axis ( G’,G” ) and the co-localization with Dlx2 ( H’,H” ). ( I ) Schematic representation of gene expression profile in different cell types of the neurogenic sequence. Circular arrow indicates proliferating cells. LV: lateral ventricle, RG: radial glia, TAP: transit amplified precursor, VZ: ventricular zone, SVZ: sub-ventricular zone. D: dorsal, L: lateral, S: septal, V: ventral. Scale bars: 100 µm ( C–F ), 20 µm ( C’–F’ ), 50 µm ( G–H ).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Plasmid Preparation, Isolation, Electroporation, Microarray, Expressing, In Situ Hybridization, Immunohistochemistry, Staining, Quantitative Proteomics, Gene Expression, Sequencing, Amplification

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: Cells were analyzed 15 days after electroporation of lateral V-SVZ progenitors by Cre mRNA in WT or Vax1cKO mice. Data are shown as means ± SD, dots represent individual animals. WT: n = 12, Vax1cKO: n = 17. Figure 2—figure supplement 2—source data 1. Quantification of tdTomato+ PGC in Vax1 mutant.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Electroporation, Mutagenesis

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to determine the expression of microRNAs in Vax1-overexpressing progenitors. PCAG-Vax1 and pCX-GFP were simultaneously introduced into NSCs by electroporation of the lateral wall of postnatal P1 brains. Lateral V-SVZ was dissected out 2 days after electroporation and GFP+ cells were isolated by flow cytometry (FACS) to perform quantitative RT-PCR analysis. ( B ) Quantification of Vax1 mRNA level by qRT-PCR in control and Vax1OE conditions, normalized to beta-actin and reported in Vax1 condition as relative level to control, validating the overexpression of Vax1 after electroporation. ( C ) Quantification of miR-7 expression in both conditions. Expression level of miR-7 was normalized by invariant expression of microRNA let-7a and reported in Vax1 condition as relative level to control. Experiments in B and C were performed in triplicate, and data were obtained from ( B ) two independent biological replications or ( C ) three technical replications. ( D ) Genome browser images representing the chromosomal portions encoding the three MiR-7 loci (depicted in pink). Mir-7–1 lies within an intronic sequence of the Hnrnpk gene whereas MiR-7–2 and MiR-7b reside within intergenic sequences. Vax1-binding sites found in the upstream regulatory region of the three MiR-7 are represented by red boxes. ( E ) Model of cross-regulatory interaction between Vax1 , miR-7, and Pax6 in the lateral V-SVZ to control the number of dopaminergic neurons generated by the neural stem cells regionalized in this aspect. This model is supported by our present data and previous work where it was shown that miR-7 was required to inhibit PAX6 expression in lateral NSCs to produce the correct number of dopaminergic neurons in the postnatal OB. Here, we propose that Vax1 acts upstream of miR-7 by positively regulating its expression and consequently inhibiting PAX6. However, it is also possible that Vax1 directly represses the expression of Pax6 mRNA (dashed line) by acting on its promoter . Additionally, Vax1 is required to generate Calbindin neurons from the ventral aspect of the lateral V-SVZ. Figure 5—source data 1. Quantification of expression level of Vax1 and miR-7 in V-SVZ cells.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Expressing, Electroporation, Isolation, Flow Cytometry, Quantitative RT-PCR, Control, Over Expression, Sequencing, Binding Assay, Generated

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to target lateral V-SVZ NSCs with Cre -mRNA. Tomato+ recombined cells were isolated 2 days after electroporation. ( B ) Vax1 mRNA level quantified by RT-PCR was normalized to beta-actin and reported in Vax1cKO condition as relative level to control (WT).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Isolation, Electroporation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet:

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Sequencing, Recombinant, Plasmid Preparation, SYBR Green Assay, Software, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TL1A Enhances the Activation of MAIT Cells Suboptimally Stimulated with IL-12 and IL-18 CD8 + T cells were enriched from healthy peripheral blood mononuclear cells (PBMCs) and stimulated overnight with different combinations of cytokines: IL-12 at 2 ng/mL, IL-18 at 50 ng/mL, IL-15 at 25 ng/mL, and TL1A from 0.01 to 100 ng/mL as indicated. (A–C) Proportions of CD8 + MAIT/CD161 + or CD161 − cells producing IFN-γ (A), TNF-α (B), or CD69 (C) following overnight stimulation with suboptimal concentrations of IL-12 and IL-18, plus varying concentrations of TL1A. (D) Representative histograms showing the expression of IFN-γ, TNF-α, GrB, and CD69 by MAIT cells after stimulation with different combinations of cytokines. (E–H) Frequency of MAIT cells expressing IFN-γ (E), TNF-α (F), GrB (G), and CD69 (H) upon stimulation with the indicated cytokines. Data were acquired from seven donors in 2–3 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Activation Assay, Expressing, Comparison

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TCR-Mediated Activation of MAIT Cells Leads to the Expression of Tissue-Repair-Associated Molecules and Accelerates Wound Healing (A–C) Gene set enrichment summary plots for stimulated sorted MAIT cell-versus-unstimulated cell-ranked genes. Depicted are the individual plots for TCR-stimulated versus UT in (A), TC-stimulated versus UT in (B), and C versus unstimulated in (C). Non-significant for C versus UT, normalized enrichment score (NES) = 1.63; p < 0.0002 for TCR versus UT, NES = 1.57; and p < 0.0002 for TC versus UT. Data were acquired from three donors in one experiment. (D) Flow cytometry analysis of the expression of TNF-α, furin, and CCL3 by CD161 ++ /MAIT CD8 + T cells in response to fixed E. coli presented by THP1 cells in the presence or absence of an anti-MR1 (αMR1) blocking antibody at the 72-h time point. (E) Statistical analysis of the expression of the effector molecules shown in (D). (F) Caco2 cells were grown to confluency and scratched with a WoundMaker device to perform in vitro wound-healing assays. Cells were supplemented with different supernatants collected from 72-h cocultures of enriched CD8 T cells with E. coli -loaded THP1 cells in the presence or absence of αMR1, as indicated. The open wound areas were quantified as percentages of the initial wound size in the Caco2 cultures. Data points are mean ± SEM and were acquired from five biological replicates in two experiments. (G) Representative pictures of the closure of the wounds in Caco2 cultures treated as in (F) were assessed with time-lapse imaging over a time course of 36 h. Data were acquired from seven donors in three experiments. Differences among conditions were analyzed by two-way ANOVA. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.001. Scale bars, 250 μm. See also Figure S5 and .

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Activation Assay, Expressing, Flow Cytometry, Blocking Assay, In Vitro, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: MAIT Cells Can Be Found Close to and within the Colonic Epithelium (A–G) Representative images showing the expression of Va7.2, CD161, CD8, PLZF, CD3, and CD103 in the lamina propria and the epithelium of fixed samples of colonic polyp tissue. Samples were mounted on cytometer chips and iteratively stained with sets of three directly fluorochrome-conjugated antibodies as described in the methods section. Depicted are a merged picture (A) and all the individual stains for Va7.2 (B), CD161(C), CD8 (D), PLZF (E), CD3 (F), and CD103 (G). White arrows mark cells showing co-expression of Va7.2, CD161, PLZF, and CD3 that were defined as MAIT cells here. Note that while CD8 was co-expressed in most of them, CD8− MAITs (arrow + asterisk) could also be found. In contrast, CD103 was rarely co-expressed on MAITs (arrow + diamond). During the iterative staining process dust particles and other detritus can be picked up by the solution flowing over the tissue creating autofluorescent artifacts (1–4). While some of these get washed away after completion of the staining cycle (1, 4), others present during multiple imaging rounds (2, 3). Scale bars, 50μm.

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Expressing, Cytometry, Staining, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet:

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Virus, Recombinant, Reverse Transcription, Activation Assay, Staining, Software

Journal: Cancer research

Article Title: Therapy-educated mesenchymal stem cells enrich for tumor initiating cells

doi: 10.1158/0008-5472.CAN-17-1547

Figure Lengend Snippet: (A-C) Eight-to-ten week old SCID mice were subcutaneously implanted with PANC1 or A549 cells (n=5 mice/group). When tumors reached 500mm3, treatment with gemcitabine (500mg/kg) or vehicle control was initiated. After 72 hours the tumors were harvested; half of the tumor was sectioned and the other half was prepared as a single cell suspension. (A) Tumor sections were immunostained using antibodies against CD105 (yellow) and αSMA (green) to identify MSCs, and CD133 (red) to identify TICs. Nuclei were stained with DAPI (blue). Red arrows represent TICs whereas white arrows represent MSCs. Scale bar, 100 µm. (B) The distance between MSCs and TICs was measured and plotted (n>15 fields/group). (C) The percentage of MSCs in tumor single cell suspensions was quantified by flow cytometry. (D-E) Eight-to-ten week old SCID mice were co-implanted with PANC1 (5x106 cells) and human MSCs (5x105 cells). (D) Tumor growth was assessed regularly. Error bars represent SE. (E) At end point, tumors were removed and prepared as single cell suspensions. The percentage of TICs was assessed by flow cytometry. *p<0.05, **p<0.01, as assessed by student t-test or one way ANOVA followed by Tukey post-hoc test (when comparing between more than two groups).

Article Snippet: To identify human pancreatic TICs, sections were stained with PE-conjugated antibodies against human prominin-1 (CD133, 1:250, Macs MiltenyiBiotec).

Techniques: Staining, Flow Cytometry

Journal: Cancer research

Article Title: Therapy-educated mesenchymal stem cells enrich for tumor initiating cells

doi: 10.1158/0008-5472.CAN-17-1547

Figure Lengend Snippet: (A) PANC1 and A549 cells were cultured in serum-free medium supplemented with conditioned medium (CM) derived from control or gemcitabine-educated MSC cultures. After 72 hours, the percentage of TICs was assessed by flow cytometry. (B) Aldehyde dehydrogenase (ALDH) activity was evaluated in PANC1 cells treated as in (A). DEAB was used as a negative control. (C) SP assay was assessed on PANC1 cells treated as in (A). (D) PANC1 and A549 cells were co-cultured with MSCs in 1:10 ratio in serum-free conditions for 72 hours. The cultures were then treated with gemcitabine (10 nM) or vehicle control for 72 hours and the percentage of TICs (CD133+) was evaluated by flow cytometry. (E) Standard and TIC-enriched PANC1 cultures were incubated with naïve or gemcitabine-educated MSCs. After 24 or 72 hours, the percentage of apoptotic cells in each culture was assessed by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, as assessed by student t-test or one way ANOVA followed by Tukey post-hoc test (when comparing between more than two groups).

Article Snippet: To identify human pancreatic TICs, sections were stained with PE-conjugated antibodies against human prominin-1 (CD133, 1:250, Macs MiltenyiBiotec).

Techniques: Cell Culture, Derivative Assay, Flow Cytometry, Activity Assay, Negative Control, Incubation

Journal: Cancer research

Article Title: Therapy-educated mesenchymal stem cells enrich for tumor initiating cells

doi: 10.1158/0008-5472.CAN-17-1547

Figure Lengend Snippet: (A) PANC1 cells were cultured in serum-free medium supplemented as follows: unsupplemented (control); MSC-derived nano-ghosts (NG); and CM derived from gemcitabine-educated MSCs (MSC-GEM). After 3 days, the percentage of TICs in each culture was evaluated by flow cytometry. (B) Eight-to-ten week old SCID mice were orthotopically implanted with PANC1 cells (5x105 cells/mouse) into the pancreas. After 4 weeks, the mice were treated with gemcitabine (500mg/kg) or vehicle control. Twenty-four hours later, mice were intravenously injected with DiO-labelled NGs. After 1 week, mice were sacrificed and tumors were sectioned. Tumor sections were immunostained with antibodies against CD133 to detect TICs (red). Nuclei were stained with DAPI (blue). The mean fluorescence intensity (MFI) of the DiO signal (green) was calculated. Scale bar, 100 µm. (C) PANC1 cells were cultured in serum-free medium supplemented as follows: unsupplemented (control), conditioned medium from untreated MSCs (MSC), CM from gemcitabine-educated MSCs (CM-MSC-GEM) alone or in combination with MSC-derived nano-ghosts (NG), AMG487-loaded NGs (NG-AMG487), or free AMG487 (1µM). After 3 days, the percentage of TICs in each culture was evaluated by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, as assessed by student t-test or one way ANOVA followed by Tukey post-hoc test (when comparing between more than two groups).

Article Snippet: To identify human pancreatic TICs, sections were stained with PE-conjugated antibodies against human prominin-1 (CD133, 1:250, Macs MiltenyiBiotec).

Techniques: Cell Culture, Derivative Assay, Flow Cytometry, Injection, Staining, Fluorescence

Journal: Cancer research

Article Title: Therapy-educated mesenchymal stem cells enrich for tumor initiating cells

doi: 10.1158/0008-5472.CAN-17-1547

Figure Lengend Snippet: (A-D) Eight-to-ten week old SCID mice were orthotopically implanted with PANC1 cells (5x105 cells/mouse). After two weeks, mice were injected with gemcitabine chemotherapy (GEM, 500mg/kg), AMG487-loaded NG (NG-AMG487) or a combination of the two. Gemcitabine was injected weekly one day before the injection of NGs over a 3 week period (white arrows). Note, mouse 3 and 4 in control group week 6 exchanged position. (A) Tumor growth was assessed using the IVIS imaging system. (B-C) Shown are plots of bioluminescence levels. Black arrows represent gemcitabine injections and red arrows represent NG-AMG487 injections. Error bars represent SE. (D) Tumor size at 6 weeks was also analyzed by micro-ultrasound. (E) At end point, tumors were removed. Half of each tumor was sectioned and the other half was prepared as a single cell suspension. Tumor sections were immunostained using antibodies against CD133 to detect TICs (green). Apoptotic cells were detected by TUNEL staining (red). Nuclei were stained with DAPI (blue). Scale bar, 100 µm. (F) The percentage of apoptotic TICs in single cell suspensions was evaluated by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, as assessed by one way ANOVA followed by Tukey post-hoc test (when comparing between more than two groups).

Article Snippet: To identify human pancreatic TICs, sections were stained with PE-conjugated antibodies against human prominin-1 (CD133, 1:250, Macs MiltenyiBiotec).

Techniques: Injection, Imaging, Micro-Ultrasound, TUNEL Assay, Staining, Flow Cytometry