Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 1. S180A mutation in CD44 prevents CS addition and results in constitutive HA binding in Jurkat cells. A, CD44 expression and HA binding were analyzed by flow cytometry in transfected Jurkat cells either unstimulated or stimulated for 8 h with PMA. The two left panels show expression levels of CD44, detected using Alexa 488 conjugated IM7, whereas the two right panels show binding to Fl-HA. B, CD44 immuno- precipitated from sodium [35S]sulfate-labeled cells grown in the presence or absence of -D-xyloside and resolved by SDS-PAGE. C, Relative quan- titation of sulfate incorporation by CD44 before and after digestion with chondroitinase ABC (ABC) or heparitinase (Hep) as measured by densi- tometry. Sulfate incorporation by untreated wild-type CD44 was set to 100% after adjustment for the level of CD44. Data are shown as the mean SD of three experiments with significance determined by the Stu- dent’s t test (, p 0.001). D, Detection of CS on immunoprecipitated CD44 by Western blotting with the anti-CS mAb 2B6. CD44 loading levels were determined by blotting with the mAb 3G12.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Binding Assay, Expressing, Cytometry, Transfection, Labeling, SDS Page, Immunoprecipitation, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 2. Cell death is preferentially induced in TCR- or PMA-stim- ulated Jurkat cells expressing a high HA binding form of CD44. A, Time course showing percentage of live cells after stimulation with the anti-TCR mAb C305. Data are shown as the mean SD of three experiments and were normalized by setting the number of live events in the untreated samples to 100%. B, Representative experiment showing the percentage of cells that were unstained (viable), single positive for Annexin V, or double positive for both Annexin V and PI following anti-TCR stimu- lation. C, Time course of cell viability during PMA stimulation as de- termined by lack of Annexin V-FITC and PI staining. Data are shown as the mean SD of three experiments. D, Same as B, except cells were stimulated with PMA.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 3. CD44 mAbs can induce or prevent cell death of transfected Jurkat cells during PMA stimulation. A, Representative experiment show- ing the percentage of cells that were viable, single positive for Annexin V, or double positive for both Annexin V and PI following incubation with Hermes-1 or Hermes-3 for 20 min and cross-linking with secondary Ab for 16 h. B, Same as A, except unstimulated or PMA stimulated cells were grown in AIMV serum-free media before coincubation of Hermes-1 and secondary Ab for 8 h. C, Graph showing percentage of viable cells fol- lowing PMA stimulation for 16 h in the presence or absence of the HA blocking anti-CD44 mAb Hermes-1 or the nonblocking anti-CD44 mAb Hermes-3. Data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.01, , p 0.001).

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Transfection, Incubation, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 4. Enhanced AICD in Jurkat transfectants is dependent upon HA binding by CD44. A, Graph showing cell viability in S180A-CD44 cells following PMA stimulation for 16 h in RPMI 1640 supplemented with 10% FCS or in RPMI 1640 with or without 5 g/ml HA. Hyaluron- idase (HA’ase) treatment was done for 30 min before PMA stimulation. Data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.05, , p 0.01, , p 0.001). B, Cell viability is shown following PMA stimulation for 16 h in AIMV serum-free media with various concentrations of HA. The HA- blocking anti-CD44 mAb Hermes-1 was added to some samples. C, The mean fluorescence intensity (MFI) for CD44 and S180A-CD44 expression (top panel) and Fl-HA binding (middle panel) at various time points during PMA stimulation is shown. The bottom panel shows the relationship be- tween Fl-HA binding and the level of CD44 expression for CD44 and S180A-CD44. Data in B and C are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Binding Assay, Blocking Assay, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 5. The size of HA affects its ability to induce cell death in PMA-stimulated cells. A, Visualization of high, intermediate (int), or low molecular mass HA by silver staining samples run on a 15% acrylamide gel. Bromophenol blue (BPB) was used as a marker dye. B, Competition assay between Fl-HA and increasing amounts of unlabeled high, interme- diate, and low molecular mass HA in S180A-CD44 cells. C, Cell viability is shown in S180A-CD44 cells following PMA stimulation for 16 h in AIMV serum-free media with various concentrations of high, intermediate, or low molecular mass HA. The HA-blocking anti-CD44 mAb Hermes-1 was added to some samples. Data in B and C are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Silver Staining, Acrylamide Gel Assay, Marker, Competitive Binding Assay, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 6. HA can rapidly induce cell death in CD44-expressing cells after PMA activation. A, Graph showing cell viability in S180A-CD44 cells suspended in AIMV media and stimulated with PMA in the presence or absence of 500 ng/ml HA. Cells were initially stimulated for 12 h, then washed twice and incubated for an additional 4 h in the presence of PMA and/or HA. Data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.05, , p 0.001). B, Time course of cell viability following the addition of HA to S180A-CD44 cells prestimulated for 12 h with PMA. Hermes-1 mAb was added 20 min before the addition of HA for one sample set. Data are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 7. HA-induced cell death does not occur via the Fas/FasL pathway. A, Cell viability was assessed in unstimulated cells incubated with the anti-Fas mAb 7C11 for various times. Data are shown as the mean SD of three experiments. B, Fas expression as determined by flow cytometry in cells cultured in 10% FCS and either stimulated with PMA for 8 h or left untreated (NT). C, FasL mRNA expression in AIMV-cultured S180A-CD44 cells as determined by semiquantitative PCR following PMA stimulation in the presence or absence of HA. -actin was used as a loading control. D, Cell viability of S180A-CD44 transfectants following the addition of HA for 2 h to cells prestimulated with PMA. Cells were preincubated for 20 min with the Fas-blocking mAb ZB4 or the FasL-blocking mAb NOK-1 before the addition of HA. E, Cell viability of S180A-CD44 cells activated for 16 h with immobilized anti-CD3 mAb OKT3 in the presence of various combinations of HA, ZB4, or Hermes-1. F, Western blot analysis of caspase 8 activation following the addition of HA to PMA-stimulated S180A-CD44 cells. Incubation of unstimulated cells with the anti-Fas mAb 7C11 for 2 h was used as a positive control. G, Same as D, except cells were preincubated for 20 min with 25 or 50 M of the pan-caspase inhibitor z-VAD-fmk. As a control, unstimulated cells were incubated with z-VAD-fmk before the addition of the 7C11 mAb. For D, E, and G data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.01, , p 0.001).

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Incubation, Expressing, Cytometry, Cell Culture, Control, Blocking Assay, Western Blot, Activation Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 8. HA-induced cell death does not occur via the mitochon- drial pathway. A, Cell viability of unstimulated Jurkat transfectants in- cubated with staurosporine for 1–3 h (left panel) or incubated in RPMI 1640 without FCS for 1–3 days (right panel). B, Time course of mito- chondrial depolarization in S180A-CD44 cells. PMA-stimulated cells were incubated with either HA or Hermes-1 mAb and goat anti-rat Ab (2°) and then labeled with the mitochondrial membrane-specific dye JC-1. Incubation of unstimulated cells with the 7C11 mAb was used as a positive control. C, Same as B, except cells were analyzed after 2 h with the membrane dye DiOC6(3) and the percentage of live depolar- ized cells (PI negative, DiOC6(3) low) is shown. D, Western blot anal- ysis of caspase 3 activation following the addition of HA to PMA- stimulated S180A-CD44 cells. Data from A, B, and C are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Incubation, Labeling, Membrane, Positive Control, Western Blot, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 10. AICD in ex vivo activated murine splenic T cells is en- hanced by the presence of HA. A, Representative experiment showing the percentage of live day 6 splenic T cells from wild-type (WT) and CD44 knockout (KO) mice following reactivation with immobilized anti-CD3 mAb 145-2C11 for 24 h (see Materials and Methods for details). Some samples were incubated with 500 ng/ml HA or with both immobilized anti-CD3 and anti-CD44 (IM7). The mean of three experiments is shown. B, Analysis of CD44 expression and Fl-HA binding of day 6 activated splenic T cells either unstimulated (thin line) or restimulated (thick line) for 24 h. The cells alone negative control (shaded) is also shown. C, Analysis of PS exposure and Fl-HA binding in day 6 wild-type splenic T cells. After restimulation for 24 h with 2.5 g/ml of immobilized anti-CD3 mAb, cells were incubated with Fl-HA for 30 min at 37°C, labeled with Annexin V-PE on ice, and then analyzed by flow cytometry. Live cells were divided into non-, low-, and high-HA binding populations and analyzed for levels of Annexin V-PE binding. The percent of cells positive for Annexin V within each population is indicated. Mean fluorescence intensity (MFI) was nor- malized between experiments by setting the intensity of the non-HA bind- ing population to 1, and data are shown as the mean SD of three ex- periments with significance determined by the Student’s t test (, p 0.05). D, Graph showing the relative decrease in cell viability in day 6 T cells incubated in the presence of 1 g/ml of the Fas blocking mAb MFL3 for 24 h on immobilized anti-CD3 mAb. HA at 500 ng/ml was added to the cells for 0.5 h, 2 h, or 24 h. To normalize between experiments, the per- centage of loss of cell viability between cells stimulated in the absence vs the presence of HA is shown. Data are the mean SEM of four experi- ments with pools of two mice per experiment. Significance (, p 0.01) is shown compared with CD44 knockout (KO) cells. E, Graph showing the

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Ex Vivo, Knock-Out, Incubation, Expressing, Binding Assay, Negative Control, Labeling, Cytometry, Blocking Assay

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: BCG infectious dose correlates with increased expression of the T-cell activation markers CD44, IL-2Rα, and IL-2Rβ. Unfractionated splenocytes (5 × 105 cells/tube) from each type of spleen were stained with FITC-labeled anti-mouse CD44, anti-mouse CD25/IL-2Rα, or anti-mouse CD122/IL-2Rβ and PE-labeled anti-mouse CD4 or anti-mouse CD8α. The percentages of CD4+ CD44hi, IL-2Rαhi, or IL-2Rβhi CD4+ (white bars) or CD8+ cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05; ∗∗, P < 0.01. Purified CD4+ T cells from the spleens of BCG HD-infected mice (day 16) were stained with FITC anti-mouse CD44 and were sorted in an EPICS Elite ESP (Beckman Coulter) into CD44low (naïve) and CD44hi (memory/effector) populations. Sorted cells were cocultured at 105 cells/well either with bone marrow-derived dendritic cells (5 × 104 cells/well) in the presence or absence of SEB (10 μg/ml) or with M12 B-lymphoma cells (5 × 104 cells/well) in the presence or absence of BCG-Ag (10 μg/ml). IFN-γ levels were determined as described in Materials and Methods. Mean ± standard deviation of triplicate wells is shown.

Article Snippet: For flow cytometry analysis, splenocytes (5 × 10 5 ) were stained with all or some of the following MAbs (all at 1:100 dilutions in R10A): fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse Vβ8 TCR (F23.1), FITC-conjugated rat anti-mouse CD25 (IL-2 Rα; 7D4), FITC-conjugated rat anti-mouse CD122 (IL-2 Rβ; TM-β1), FITC-conjugated rat anti-mouse I-A d /I-E d (2G9) (all from PharMingen), or FITC-conjugated rat anti-mouse CD44 (IM7.8.1; Cedarlane Laboratories, Hornby, Canada).

Techniques: Expressing, Activation Assay, Staining, Labeling, Flow Cytometry, Purification, Infection, Derivative Assay, Standard Deviation

Journal: The American Journal of Pathology

Article Title: Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

doi: 10.1016/j.ajpath.2014.08.029

Figure Lengend Snippet: Primary Antibodies Used in This Study

Article Snippet: All fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and used at a dilution of 1:200: anti-mouse IgG (DyLight 549), number 715-505-151, subclass 1; anti-mouse IgG (cy2 conjugated, number 115-225-205), subclass 1; anti-mouse IgG (cy3 conjugated, number 115-165-206), subclass 2a; and anti-rabbit IgG (cy2 conjugated, number 715-165-151, and DyLight 488, number 715-095-151). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Isotype Company or producer Catalog no. Dilution Mouse anti-KRT18 IgG1 Abcam (Cambridge, MA) Ab668 1:200 Rabbit anti-KRT18 Polyclonal Novus Biologicals, LLC (Littleton, CO) NBP1-67610 1:150 Rabbit anti-human CLDN 7 Polyclonal Abcam Ab27487 1:100 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC NB-600-308 1:1000 Rabbit anti-rat Desmin Polyclonal AnaSpec, Inc. (San Jose, CA) 29593 1:200 Mouse anti-rat THY1/CD90 IgG1 Cedarlane (Hornby, ON, Canada) CL005AP 1:50 Mouse anti-rat THY1/CD90 IgG1 BD Biosciences (San Jose, CA) 553016 1:50 Mouse anti-rat THY1/CD90 IgG2a Antibody Solutions (Sunnyvale, CA) Ab53226 1:25 Mouse anti-CDH1 IgG2a Transduction Lab, BD Biosciences 61081 1:50 Mouse anti-CDH1 IgG1 ECM Biosciences (Versailles, KY) CM1681 1:50 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC AB-4 1:500 Mouse anti-rat CD44 IgG2a Cedarlane CL044 1:50 Mouse OV6 IgG1 Kind gift from Dr. Stewart Sell (Wadsworth Center, Albany, NY) NA 1:50 Mouse anti-ACTA2 IgG2a Abcam Ab11003 1:100 Mouse anti-GFAP IgG1 Abcam Ab80842 1:400 Mouse anti-rat VCAM1 IgG1 Bio Legend (San Diego, CA) 200401 1:100 Rabbit anti-CD29/integrin B1 Monoclonal Epitomics (Burlingame, CA) 2288-1 1:50 Mouse anti-vimentin IgG1 Lab Vision Corporation (Fremont, CA) MS129-PO 1:50 Mouse anti-rat NT5E IgG1 BD Biosciences 551123 1:50 Open in a separate window NA, not applicable.

Techniques: Transduction

Journal: The American Journal of Pathology

Article Title: Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

doi: 10.1016/j.ajpath.2014.08.029

Figure Lengend Snippet: Primers Used in This Study

Article Snippet: All fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and used at a dilution of 1:200: anti-mouse IgG (DyLight 549), number 715-505-151, subclass 1; anti-mouse IgG (cy2 conjugated, number 115-225-205), subclass 1; anti-mouse IgG (cy3 conjugated, number 115-165-206), subclass 2a; and anti-rabbit IgG (cy2 conjugated, number 715-165-151, and DyLight 488, number 715-095-151). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Isotype Company or producer Catalog no. Dilution Mouse anti-KRT18 IgG1 Abcam (Cambridge, MA) Ab668 1:200 Rabbit anti-KRT18 Polyclonal Novus Biologicals, LLC (Littleton, CO) NBP1-67610 1:150 Rabbit anti-human CLDN 7 Polyclonal Abcam Ab27487 1:100 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC NB-600-308 1:1000 Rabbit anti-rat Desmin Polyclonal AnaSpec, Inc. (San Jose, CA) 29593 1:200 Mouse anti-rat THY1/CD90 IgG1 Cedarlane (Hornby, ON, Canada) CL005AP 1:50 Mouse anti-rat THY1/CD90 IgG1 BD Biosciences (San Jose, CA) 553016 1:50 Mouse anti-rat THY1/CD90 IgG2a Antibody Solutions (Sunnyvale, CA) Ab53226 1:25 Mouse anti-CDH1 IgG2a Transduction Lab, BD Biosciences 61081 1:50 Mouse anti-CDH1 IgG1 ECM Biosciences (Versailles, KY) CM1681 1:50 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC AB-4 1:500 Mouse anti-rat CD44 IgG2a Cedarlane CL044 1:50 Mouse OV6 IgG1 Kind gift from Dr. Stewart Sell (Wadsworth Center, Albany, NY) NA 1:50 Mouse anti-ACTA2 IgG2a Abcam Ab11003 1:100 Mouse anti-GFAP IgG1 Abcam Ab80842 1:400 Mouse anti-rat VCAM1 IgG1 Bio Legend (San Diego, CA) 200401 1:100 Rabbit anti-CD29/integrin B1 Monoclonal Epitomics (Burlingame, CA) 2288-1 1:50 Mouse anti-vimentin IgG1 Lab Vision Corporation (Fremont, CA) MS129-PO 1:50 Mouse anti-rat NT5E IgG1 BD Biosciences 551123 1:50 Open in a separate window NA, not applicable.

Techniques: Sequencing, Amplification

Journal: The American Journal of Pathology

Article Title: Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

doi: 10.1016/j.ajpath.2014.08.029

Figure Lengend Snippet: Immunofluorescence microscopy analysis for expression of mesenchymal and epithelial cell marker proteins in 13-11-3-2 cell line. Mesenchymal cell markers were as follows: vascular cell adhesion molecule 1 (VCAM; A); Desmin (B); ACTA2 (α smooth muscle actin; C); glial fibrillary acidic protein (GFAP; F); Vimentin (G); Thy1 (CD90; H); CD73 (Ecto-5′-Nucleotidase; K); and CD29 (Integrin β1; L); Epithelial cell markers were as follows: KRT18 (Cytokeratin 18; D); OV-6 (E); CD44 (I); CDH1 (epithelial cadherin; J); CLDN7 (Claudin-7; M); phase-contrast microphotograph of cultured 13-11-3-5 cells (N); and phase-contrast microphotograph of cultured 13-11-3-3 cells (O). Original magnifications: ×40 (M); ×10 (N and O).

Article Snippet: All fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and used at a dilution of 1:200: anti-mouse IgG (DyLight 549), number 715-505-151, subclass 1; anti-mouse IgG (cy2 conjugated, number 115-225-205), subclass 1; anti-mouse IgG (cy3 conjugated, number 115-165-206), subclass 2a; and anti-rabbit IgG (cy2 conjugated, number 715-165-151, and DyLight 488, number 715-095-151). table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies Isotype Company or producer Catalog no. Dilution Mouse anti-KRT18 IgG1 Abcam (Cambridge, MA) Ab668 1:200 Rabbit anti-KRT18 Polyclonal Novus Biologicals, LLC (Littleton, CO) NBP1-67610 1:150 Rabbit anti-human CLDN 7 Polyclonal Abcam Ab27487 1:100 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC NB-600-308 1:1000 Rabbit anti-rat Desmin Polyclonal AnaSpec, Inc. (San Jose, CA) 29593 1:200 Mouse anti-rat THY1/CD90 IgG1 Cedarlane (Hornby, ON, Canada) CL005AP 1:50 Mouse anti-rat THY1/CD90 IgG1 BD Biosciences (San Jose, CA) 553016 1:50 Mouse anti-rat THY1/CD90 IgG2a Antibody Solutions (Sunnyvale, CA) Ab53226 1:25 Mouse anti-CDH1 IgG2a Transduction Lab, BD Biosciences 61081 1:50 Mouse anti-CDH1 IgG1 ECM Biosciences (Versailles, KY) CM1681 1:50 Rabbit anti-GFP Polyclonal Novus Biologicals, LLC AB-4 1:500 Mouse anti-rat CD44 IgG2a Cedarlane CL044 1:50 Mouse OV6 IgG1 Kind gift from Dr. Stewart Sell (Wadsworth Center, Albany, NY) NA 1:50 Mouse anti-ACTA2 IgG2a Abcam Ab11003 1:100 Mouse anti-GFAP IgG1 Abcam Ab80842 1:400 Mouse anti-rat VCAM1 IgG1 Bio Legend (San Diego, CA) 200401 1:100 Rabbit anti-CD29/integrin B1 Monoclonal Epitomics (Burlingame, CA) 2288-1 1:50 Mouse anti-vimentin IgG1 Lab Vision Corporation (Fremont, CA) MS129-PO 1:50 Mouse anti-rat NT5E IgG1 BD Biosciences 551123 1:50 Open in a separate window NA, not applicable.

Techniques: Immunofluorescence, Microscopy, Expressing, Marker, Cell Culture

Journal:

Article Title: HIV-specific cytotoxic T lymphocytes traffic to lymph nodes and localize at sites of HIV replication and cell death

doi:

Figure Lengend Snippet: Representative histologic sections of inguinal lymph node collected from patient 2 by excisional biopsy 4 days after the second infusion of neo-modified HIV-specific CTL. Serial sections were analyzed for sites of localization of neo-modified CD8+ CTL (PCR-ISH) (a), cells expressing granzyme-B (anti-GrB mAb) (b), cells expressing HIV tat fusion transcripts (RT-PCR-ISH) (c), cells expressing the CC-chemokine MIP-1β (anti-human MIP-1β mAb) (d), and cells showing DNA fragmentation (TUNEL “apoptosis” assay) (e). Dual stains were used to identify the proportion of CD4+ (anti-CD4 mAb + TUNEL) (f; inset shows higher magnification of dual-stained cells [arrows]) and CD8+ (anti-CD8 mAb + TUNEL) (g) T lymphocytes demonstrating apoptosis. The HIV-specific CTL expressed the cell surface receptors CD8 and CCR5 and could be stimulated to express granzyme-B before their infusion (h: anti-CD8 mAb (left), anti-CCR5 mAb (middle), anti-GrB mAb (right). Antibody labels were detected by immunoperoxidase staining with DAB as the chromogen and apoptotic cells detected by TUNEL, followed by anti-DIG mAb, and developed with BCIP/NBT. f, follicular germinal centers. Bar, 100 μm.

Article Snippet: Immunostains were also performed following standard procedure ( 9 , 19 , 26 ), which included an Ag-retrieval step (steaming in 1 mM citrate buffer, pH 6.0, for 20 minutes) and used mAb’s (20 μg/mL) to the human CD8 (clone C8/144B, IgG 1 ; DAKO Corporation, Carpinteria, California, USA); human CCR5 (clone 45531.111, IgG2b; Endogen Inc., Woburn, Massachusetts, USA); human MIP-1α (clone 11A3, IgG 2a ; LeukoSite Inc., Cambridge, Massachusetts, USA); human MIP-1β (clone 1F12, IgG 2b ; LeukoSite Inc.); and granzyme-B (clone B18.1, IgG 1 ; Alexis Corp., San Diego, California, USA), a serine protease stored in granules of activated CTL.

Techniques: Modification, Expressing, Reverse Transcription Polymerase Chain Reaction, TUNEL Assay, Apoptosis Assay, Staining, Immunoperoxidase Staining