Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: α5β1 and αv integrin-specific antibodies block cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation induced by hMPV F protein expression in the presence of integrin antibodies. Vero E6 cells in 48-well plates were transfected with 0.8 μg of pCAGGS-F or pCAGGS. After incubation with a plasmid-Lipofectamine mixture for 8 h, cells continued to grow in Opti-MEM containing 10 μg/ml of integrin antibody and 0.2 μg/ml of TPCK-trypsin. At 48 h, monolayers were fixed with methanol and stained with Giemsa. Syncytia are indicated by arrows. (B) Content-mixing fusion assay in the presence of integrin antibodies. Vero E6 cells were cotransfected with 2 μg of pCAGGS-F and a reporter gene plasmid (pGINT7). At 24 h posttransfection, the cells were detached with trypsin and mixed with equal numbers of BHK-SR19-T7 cells, which express T7 RNA polymerase. Then, the cells were incubated with 2 ml of Opti-MEM containing 5 μg/ml of selected integrin antibody and 0.2 μg/ml of TPCK-trypsin for 12 h. The cells were lysed and mixed with an equal amount of the β-galactosidase substrate chlorophenol red-β-d-galactopyranoside (16 mM). The extent of fusion was quantitated by use of a microplate spectrophotometer at an absorbance of 570 nm. Percent fusion for each antibody treatment was normalized to the fusion of pCAGGS-F in the absence of integrin antibody. The data shown are averages for three independent experiments.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Blocking Assay, Expressing, Transfection, Incubation, Plasmid Preparation, Staining, Single Vesicle Fusion Assay, Spectrophotometry

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Knockdown, Expressing, Control, Transfection, Western Blot, Staining, Quantitation Assay, Microscopy

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: Integrin α5β1and αv antibodies inhibit hMPV infectivity in host cells. Confluent monolayers of LLC-MK2 cells in 96-well plates were pretreated with each integrin antibody (20 μg/ml) at 37°C for 1 h. Then, the cells were shifted to a 4°C incubator for 30 min. The cells were incubated with hMPV at an MOI of 100 PFU/well. After incubation on ice for 1 h (with shaking every 15 min), the inoculum was removed and the cells were washed with cold Opti-MEM 3 times. The infected cells were then incubated with fresh DMEM at 37°C in 5% CO2. After 24 h, the binding and infectivity were determined by counting the number of immunostaining spots. The percent infectivity for each antibody treatment was normalized by the infectivity of hMPV without antibody treatment. The data shown are averages for three independent experiments.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Infection, Incubation, Binding Assay, Immunostaining

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: Infectivity of hMPV in an integrin-deficient cell line. (A) hMPV has defects in infectivity in an integrin-deficient cell line. Confluent monolayers of GD1286 or GD25 cells in 24-well plates were incubated at 4°C for 30 min. The cells were infected with 100 PFU of hMPV per well. After incubation on ice for 1 h (with shaking every 15 min), the inoculum was removed and the cells were washed with cold DMEM 3 times. The infected cells were then incubated with fresh medium at 37°C in 5% CO2. After 24 h, the binding capacity was determined by counting the number of immunostaining spots. Percent infectivity in GD25 cells was normalized by the infectivity of hMPV in GD1286 cells. The data shown are averages for three independent experiments. (B) hMPV forms much smaller immunospots in an integrin-deficient cell line. Confluent monolayers of GD1286 or GD25 cells were infected with hMPV. After 24 h, immunostaining assay was performed, and immunospots formed by hMPV were visualized.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Infection, Incubation, Binding Assay, Immunostaining

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: Location of the RGD motif in the predicted structure of hMPV F protein. (A) Predicted hMPV F-protein monomer. The structure was predicted using the Modeler (version 9.0) program on the basis of the prefusion crystal structure of RSV F protein (PDB accession no. 4JHW) as the template. The putative integrin-binding sites (R329 and D331) are highlighted. (B) Predicted hMPV F-protein trimer. The surface of each monomer in the F-protein trimer is highlighted in a different color. The RGD motif is located on the contact region of each subunit of the F-protein trimer. (C) Location of the RGD motif in hMPV F-protein monomer. The partial structure of hMPV F protein containing DI, DII, and DIII was solved (PDB accession no. 4DAG) (49). The location of the RGD motif is highlighted. (D) The location of the RGD motif in hMPV F-protein trimer. The surface of each monomer in the F-protein trimer is highlighted in a different color.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Binding Assay

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Diarrheagenic E. coli bind to integrin α 5 β 1. (a) EAEC, EHEC, and ETEC were added to integrin α 5 β 1- and BSA-coated wells and bacterial binding was detected by ELISA, using anti-O44, anti-O157, and anti-O78 serum, respectively. (b) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells. EAEC binding was detected by ELISA, using anti-O44 serum. *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Fibronectin/integrin α 5 β 1 complex increases the adhesion of EAEC strain 042. (a) Integrin α 5 β 1- and BSA-coated wells were incubated with increasing concentrations of fibronectin (25, 50, and 100 ng) or (b) with 100 ng of fibronectin (Fn) for 15, 30, or 60 min. Bound fibronectin was detected by ELISA, using anti-fibronectin antibodies. (c) EAEC 042 was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin. (d) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells incubated with fibronectin. EAEC binding was detected by ELISA using anti-O44 serum. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: AafA binding to fibronectin/integrin α 5 β 1 complex. (a) AafA- dsc protein was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin (Fn). AafA- dsc binding was detected by ELISA using anti-AafA antibodies. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05). (b) AafA- dsc and fibronectin or (c) AafA- dsc , fibronectin, and integrin α 5 β 1 were mixed and the complex formed was added to control column or a column with anti-AafA antibodies for coimmunoprecipitation analysis. The eluted fraction obtained from control column (E1 and E3) or a column with anti-AafA antibodies (E2 and E4) was analyzed by Dot blot, using anti-AafA, anti-integrin α 5, and anti-fibronectin antibodies. The resulting autoradiography was scanned and signal intensity was quantified by UN-SCAN-IT 6.1 software. One representative experiment of three independent experiments with similar result is shown.

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control, Dot Blot, Autoradiography, Software

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Adhesion of EAEC strain 042 to T84 cells. T84 cells preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn), Fn and anti-integrin α 5 β 1 (RGD), or anti-integrin α 5 β 1 ( α 5) were infected with EAEC strain 042. The number of adherent bacteria was determined by colony forming unit counts (CFU). *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Control, Infection, Bacteria

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Integrin α 5 expression knockdown reduces EAEC strain 042 fibronectin-mediated binding to epithelial cells. HEp-2 cells nontransfected and transfected with scrambled or integrin α 5 shRNA were preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn) and then infected with EAEC strain 042. Numbers of adherent bacteria were determined by colony forming unit counts (CFU). *Significantly different compared to control treatment ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Knockdown, Binding Assay, Transfection, shRNA, Control, Infection, Bacteria

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: The expression level of integrin α5 subunit in Ph+ leukemia cell line SUP-B15 is significantly increased after serum starvation . (A) The percentages of cells expressing integrin α4, α5, and β1 subunits before and after serum starvation for 7 h detected by flow cytometry. (B) The mean fluorescent intensity (MFI) of integrin α4, α5, and β1 subunits from whole alive cell population before and after serum starvation for 7 h detected by flow cytometry.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Integrin α5 subunit antibody inhibits the adhesion of Ph+ leukemia cells to human fibronectin and enhances the killing of imatinib . (A) Cell adhesion assay showed that α5 subunit inhibitory antibody (CD49e, clone IIA1 BD Biosciences) was the only tested integrin antibody that significantly inhibited the adhesion of Ph+ leukemia cells to fibronectin with adhesion percentage of 6.6 ± 3.8% compared with control IgG of 44.8 ± 7.9%, p < 0.01. (B) . Annexin-V plus PI by flow cytometry. (i) P2 gate identifies the CD38 positive cell population representing SUP-B15 Ph+ leukemia cells. (ii) Ph+ leukemia cells grown on HS-5 stromal cells. (iii) Ph+ leukemia cells treated with 10 μM imatinib (IM). (iv) SUP-B15 cultured with stromal cells treated with 10 μM imatinib. (v) SUP-B15 cultured with stromal cells treated with 10 μM imatinib and 10 μg/ml anti-α5 Ab. (C) Quantitative analysis for apoptosis rate of different conditions. *After culture on HS-5 cells for 24 h, the apoptosis rate of leukemia cells SUP-B15 decreased from 29.4 ± 2.3 to 16.7 ± 3%, p < 0.05. # When inhibitory antibody to integrin α5 was combined with imatinib to treat Ph+ leukemia cells cultured on stromal cells, the apoptosis rate 38.0 ± 8.0% was significantly increased compared with imatinib by itself 25.7 ± 3.3%, p < 0.05.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Cell Adhesion Assay, Control, Flow Cytometry, Cell Culture

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Blocking integrin α5 affects the engraftment of Ph+ leukemia cells in immunodeficient mice . (A) The incubation for 1 h of Ph+ leukemia cells with disintegrin, a peptide inhibitor of integrins, impaired the engraftment of leukemia in NSG mice. Representative figures of n = 2 showed bioluminescence imaging from day 7 to 28 after inoculation of leukemia cells. (B) Animal total body bioluminescence was measured using the Xenogen IVIS Imaging System 200 Series with total imaging time of 2 min and compared with control animals that received cells that were not treated with Echistatin. (C) Anti-integrin α5 inhibitory antibodies clone IIA1 (BD Biosciences) and clone P1D6 (Millipore) decreased the engraftment of Ph+ leukemia cells in the bone marrow of NOD/SCID mice ( n = 2).

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Blocking Assay, Incubation, Imaging, Control

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Knocking down integrin α5 delayed the engraftment of Ph+ leukemia cells in immunodeficient NOD/SCID mice . (A) Real-time QPCR assay showed that we had successfully knocked down the expression level of the integrin α5 knocking in clone 10. The cycle threshold (Ct) of the target gene (integrin α5) was normalized to the chosen reference gene GAPHD. Relative quantification, R = 2 −(ΔCt sample − ΔCt control) . Result shows the fold change normalized to SUP-LUC2 cells. (B) Western blot showed a reduced protein expression level of integrin α5 in clone 10. (C) Flow cytometry showed reduced mean fluorescent intensity of integrin α5 in clone 10. (D) Representative figures showed bioluminescent imaging at 2 weeks, and 5–8 weeks post leukemia cells inoculation. (E) Animal total body bioluminescence was quantified to compare with control. **The levels of bioluminescence became most significantly different 2 months post inoculation between the α5 knock-down group and control group [mean (SD) radiance vs. control, 5.3 (0.1) vs. 2.1 (0.2) × 10 6 p/s/cm 2 /sr, p < 0.01 at 2 months, n = 3.].

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Quantitative Proteomics, Control, Western Blot, Flow Cytometry, Imaging, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Integrin expression profiles in HT-1080 and MV3 cells and RNAi-based integrin targeting in HT-1080 cells. (A and B) Surface expression pattern of integrin β and α chains on HT-1080 (A) and β chains on MV3 (B) cells determined by flow cytometry. Black line, isotype control. Values, mean fluorescence (minus isotype values). (C–E) Downregulation of β1 and β3 integrins in HT-1080 cells by shRNA. (C) Knockdown efficiency of β1 integrin in dual-color HT-1080 cells (Western blot), compared with nontransduced (NT) and empty vector (EV)–transduced cells. β-Tubulin, loading control. (D and E) Upregulation of β3 integrins after downregulation of β1 integrin (D) and efficient downregulation of both β1 and β3 integrins after β1/β3RNAi (E) determined by flow cytometry. Surface expression pattern of β1 and β3 integrins on β1RNAi cells or β1/β3RNAi cells (red lines) compared with cells transfected with empty vector (EV, blue lines). Black line, isotype control. Values, mean fluorescence (minus isotype values). Stability of β1/β3 downregulation was routinely verified, and no outlier behavior or drift of expression to other integrin β-chains was noted (data not shown). (F) Reduction of β1 integrin adhesion epitope detected by FITC-conjugated mAb 4B4 on vector control (EV; left) and β1RNAi cells (right) after epitope saturation with unconjugated mAb 4B4 (3 µg/ml; blue line showing residual epitopes) compared with unspecific IgG1 (red line; total epitopes). Black line, isotype control staining (Iso). Values indicate mean fluorescence intensities. (G) Diminished phosphoErk signal (MAPK signaling) after β1/β3 integrin targeting (day 7). Histograms show the mean pixel fluorescence (MF) intensity of pErk from control (HT-1080 wild type) and β1/β3 integrin targeted tumors (T, dotted lines, identified by H2B-EGFP label) compared with pErk signal in the surrounding stroma (S), which further contained hair follicles (HF) with strong endogenous pErk expression. Ratio of tumor- and stroma-derived pErk intensity is displayed as medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) from one sample determined from 10 independent regions of the corresponding stroma region after exclusion of hair follicles, with a ratio of 1.0 (red dashed line) when signal intensity of both regions was equal. Calibration bar, pixel intensity. Scale bars, 100 µm (overview); 10 µm (inset).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Expressing, Flow Cytometry, Fluorescence, shRNA, Western Blot, Plasmid Preparation, Transfection, Staining, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Compromised tumor integrity and persistence of the invasion niche by RNAi- and antibody-based targeting of β1/β3 integrins in HT-1080 tumors. (A) Experimental procedure for administration of anti-β1 integrin mAb 4B4 or IgG1 and sequential intravital microscopy of the tumor response to integrin interference. Fluo, epifluorescence overview microscopy. MPM, subcellular-resolved multiphoton microscopy. (B) Time course of tumor growth or regression in control tumors transduced with empty vectors (p-puro/p-neo), β1RNAi or β1/β3RNAi in the absence or presence of IgG1 or anti-β1 integrin mAb 4B4. White arrowheads, onset of collective invasion. Numbers (right column), percentage mean regression of the tumor core (day 13 compared with day 6) from three to four independent tumors. Scale bars, 1 mm. (C) Fractions of mitotic and dead cells (day 6) quantified based on nuclear morphology for different interference schemes displayed as medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) from 20 independent fields from three to four independent tumors. Per condition, 19–20 nuclei were analyzed for the core and ∼10 nuclei for the invasion zone. *, P = 0.01; ***, P < 0.0001; ns, not significant. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.0125). (D) Tumor development in response to the indicated interference procedures. Data show the means ± SD from three to four independent tumors. *, P = 0.0286. Statistics, Mann–Whitney U test. (E) Preferential survival of invading collective strands after combined β1/β3 integrin targeting. Z-projections of the same tumor region. Black box in upper left corner (day 6) results from stitching of adjacent images without complete overlap. Insets, mitotic figures in collective strands. Arrowheads, detachment of cell groups and individualized cells. Scale bar, 250 µm. (F) Median residual volume of tumor core and collective invasion (CI) zones after β1/β3RNAi combined with mAb 4B4 (day 13 compared with day 6) from three independent tumors. *, P < 0.05. Statistics, paired t test.

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Intravital Microscopy, Microscopy, Transduction, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Radiosensitization of HT-1080 tumors by β1/β3 integrin RNA interference combined with antibody-based β1 integrin targeting. (A) Protocol for administration of anti-β1 (4B4) or IgG1 combined with fractionated IR and sequential intravital imaging of the tumor response. Fluo, epifluorescence overview microscopy. MPM, subcellular-resolved multiphoton microscopy. (B) Topology and extent of the invasion zone in response to fractionated IR combined with single-integrin (β1) or dual β1/β3 integrin interference. Epifluorescence (left) and 3D reconstructed z-projections from regions marked by dashed boxes using multiphoton microscopy (right; day 13). White asterisks, apoptotic nuclei. Scale bars, 1 mm (left); 250 µm (right). (C) Time-dependent tumor volume. Data show the means ± SD from three to four independent tumors, with P values for comparing irradiated integrin-targeted tumors to irradiated control tumors. (*), P = 0.006; *, P = 0.004. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.005). (D) Regression of tumor core and collective invasion (CI) zone after IR with or without integrin mono- or dual interference. Data show median residual areas, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of day 13 normalized to day 6. Per condition, four tumors were analyzed. (*), P = 0.03; ns, not significant. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.0125).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Imaging, Microscopy, Irradiation, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Dual-integrin targeting abrogates radioresistance in the collective invasion niche. (A) Protocol for administration of anti-β1 (4B4) and αV integrin (17E6) mAbs or IgG1 combined with fractionated IR and sequential intravital imaging of the tumor response. Fluo, epifluorescence overview microscopy. MPM, subcellular-resolved multiphoton microscopy. (B) Radiation response of tumor core and collective invasion zone after combined treatment with mAbs 4B4 and 17E6 compared with IgG1-treated control (day 13). Black box in upper right corner (HT-1080, IgG 1 ) results from stitching of adjacent images without complete overlap. Asterisks, areas of regression. Arrows, persisting invasion strands. Alexa Fluor 660–conjugated dextran-perfused blood vessels. Second harmonic generation (SHG) originates from muscle and collagen fibers. Scale bar, 250 µm. (C) Frequency of dead cells in core and collective invasion (CI) zone after antibody-based integrin targeting and/or IR (day 6). Data show the medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of ∼20 nuclei per condition and tumor regions from four to five tumors, reflecting a total of 9–28 different microscopic fields. *, P = 0.01; **, P = 0.001; ***, P < 0.0001; ns, not significant. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.0125). (D) Extent of tumor regression in core and collective invasion zone of irradiated tumors combined with or without integrin targeting. Data show median residual areas, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of day 13 normalized to day 6 from four independent tumors. *, P = 0.03. Statistics, Mann–Whitney U test.

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Imaging, Microscopy, MANN-WHITNEY, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Radiosensitization of sarcoma and melanoma tumors by antibody-based integrin interference and procedures and outcome of long-term therapy response. (A–D) Tumor morphology and quantification of radiosensitization assessed by intravital microscopy. (A and B) Time-dependent growth or regression of HT-1080 or MV3 lesions in response to the indicated treatment conditions. n.a., not analyzed due to humane endpoint after day 13 (tumor >2 cm 3 ). Images of untreated HT-1080 and MV3 tumors are also shown in Fig. S1 A. Asterisks, regression tumor core. Arrowheads, tumor remnants. Scale bars, 1 mm. (C) Time-dependent tumor volume during and after treatment with IgG1 or mAb 4B4 + 17E6 with or without IR. Data show the means ± SD from three to four (HT-1080) or three to five (MV3) independent lesions. *, P = 0.0286 (comparison IgG1/IR control with 4B4/17E6 and IR [day 15]). Statistics, Mann–Whitney U test. (D) Mitotic frequencies in nonirradiated and irradiated tumor core and collective invasion (CI) zone. Data show the medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of 10–20 nuclei per tumor region and condition from 7 to 23 independent fields from four (HT-1080) or three to five (MV3) independent tumors. ***, P < 0.0001. Statistics, Mann–Whitney U test. (E) Long-term follow-up (day 26) after treatment with 4B4 and 17E6 and IR, revealing minimal residual disease. Dotted gray line, position of former tumor. Box, position of lower panel. Zoom shows surviving cells without mitotic activity (arrowheads) and cytoplasm-free, condensed nuclei of disintegrated cells (asterisks). Scale bars, 250 µm. (F) Example tumor undergoing complete regression after therapy monitored longitudinally by whole-body fluorescence imaging. (G) IR dose escalation study for intradermal HT-1080 and MV3 tumors (window-free dermis). Left panels, overall survival after IR using the indicated doses (6–10 mice per group). Right panel, cure rate measured as percentage of tumors that did not relapse after IR. Black dashed line, IR dose with 30% cure rate (5 × 2 Gy for HT-1080 tumors, 5 × 3 Gy for MV3 tumors). (H) Dual-color detection of lung and lymph node metastases. Microscopic whole-organ screen (not depicted) was followed by analysis of cryosections (depicted). Scale bars, 100 µm (overview); 10 µm (inset). (I) Identification of minimal residual disease at the endpoint. The dorsal skin was screened from the deep fascia for presence or absence of fluorescent tumor remnants (left panel). In case of doubt, subregions were additionally sectioned for analysis by anti-EGFP immunohistochemistry (IHC; right panel). Images show typical tumor-negative outcome. Scale bar, 100 µm. (J) Examples of minimal residual lesions present at the tumor implantation site at the endpoint (day 180). Tumor remnants with strand-like pattern of green-fluorescent tumor nuclei (H2B-EGFP) followed by tissue sectioning and validation by anti-EGFP IHC. Dashed line, approximate position of tissue cross section. Arrowheads, intact H2B-EGFP–positive tumor nuclei. Right panel, validation of EGFP-positive tumor remnants and positive anti-EGFP IHC side by side. Scale bars, 100 µm (overview); 10 µm (details).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Intravital Microscopy, MANN-WHITNEY, Irradiation, Activity Assay, Fluorescence, Imaging, Immunohistochemistry, Tumor Implantation

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Dual-targeted but not individual anti-integrin therapy to enhance radiation response, tumor eradication, and long-term survival. (A) Treatment schemes for HT-1080 and MV3 tumors. Tumor cells were injected at day 0, resulting in an intradermally growing tumor located along the dorsal midline (dashed line). Example image, intradermal HT-1080 lesion. Time points of IR and antibody administration are indicated. (B) Tumor lesion (T) after implantation in imaging window–free mouse. Intradermal localization was confirmed by high-frequency ultrasound. (C) Collective invasion (CI) pattern in intradermal tumors in imaging window–free dermis (maximum-intensity projections). Number of multicellular strands per tumor was counted from 50-µm-thick tumor sections from nine (HT-1080) and seven (MV3) tumors. Scale bar, 100 µm. (D) Tumor-free overall survival of mice after application of treatment, including fractionated IR without and with individual and dual-targeted integrin inhibition with antibodies 4B4 and/or 17E6, compared with IR combined with isotypic control antibody (representing IR alone without integrin targeting). Mice were sacrificed after 180 d or earlier, upon humane endpoint criteria (tumor size of 2 cm 3 , ulceration, weight loss, or poor overall condition due to internal metastasis). See for details on mouse numbers (8–12 mice per group), metastasis formation, and tumor remnants. Gray-shaded area, therapy phase. *, P = 0.01; **, P = 0.0003; ***, P < 0.0001; ns, not significant. Statistics, log-rank survival analysis (Bonferroni-corrected thresholds: P = 0.01 [HT-1080] and P = 0.008 [MV3]).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Injection, Imaging, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Survival rates and tumor outcome for long-term follow-up experiments on integrin-targeted and IR therapy in HT-1080 sarcoma xenografts

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Survival rates and tumor outcome for long-term follow-up experiments on integrin-targeted and IR therapy in MV3 melanoma xenografts

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: