Journal: Journal for Immunotherapy of Cancer

Article Title: Systemic and intravesical adoptive cell therapy of tumor-reactive T cells can decrease bladder tumor growth in vivo

doi: 10.1136/jitc-2020-001673

Figure Lengend Snippet: Intravesical adoptive cellular therapy (ACT) using OT-I T cells delays ovalbumin expressing MB49 tumor cell line (MB49OVA) orthotopic tumor growth. Mice were inoculated with orthotopic MB49OVA tumors. Mice were treated with OT-I cells labeled with CellTrace Violet (CTV) delivered intravesically. (A) After 3 hours of instillation, tumors were collected and analyzed for CTV-labeled OT-I T cells by flow cytometry. (B) Mice with MB49OVA orthotopic tumors were treated with unlabeled OT-I T cells and monitored for tumor growth by ultrasound. ACT-treated mice had a reduction in tumor volume compared with phosphate-buffered saline (PBS)-treated mice and improved survival (C). (D) Tumors were collected on day 60 and analyzed for CD3 + , CD8 + , CD4 + and CD8 + OVA tetramer + T cells by flow cytometry. (E) Representative ultrasound images from mice in each treatment group −2 days prior to treatment and 44 days post-treatment. N=8 per group. Repeated 3 times.

Article Snippet: Samples were washed with flow buffer (1.0 L PBS, 5% heat-inactivated fetal bovine serum, 1 mM EDTA, 0.1% sodium azide) and stained for OVA tetramer (MBL International, Woburn, Massachusetts, catalog# TB5001-1) at 4°C for 20 min in the dark followed by cell surface markers CD3 (catalog# 565643), CD8 (catalog# 558106), CD4 (catalog# 550954), CXCR3 (catalog# 562152) and CD45.1 (catalog# 560578, all from BD Biosciences, San Jose, California, USA) at 4°C for 20 min in the dark.

Techniques: Expressing, Labeling, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Myosin-X is essential to the intercellular spread of HIV-1 Nef through tunneling nanotubes

doi: 10.1007/s12079-018-0493-z

Figure Lengend Snippet: CEM-T4 were mixed in a 1 to 3 ratio with Raw 264.7 cells (± CdCl2) as controls and compared to mixes with CEM-T4 and N5 (± CdCl2). The mixed population of cells were analyzed by flow cytometry for cell surface CD4 staining. Representative 2-D scatter plots of the CD4 fluorescence intensity (log scale) in CEM-T4 mixed with N5 (red) vs Raw 264.7 (black) not-treated (a) or treated with CdCl2 for 24 h (b). A downward shift in CD4 fluorescence can be observed between N5 vs Raw 264.7 treated with CdCl2. Representative Cumulative Distribution Function (CDF) (c) and histogram (d) graphs of fluorescence intensity of CD4 (log scale) in CEM-T4 in response to the various co-cultures are plotted. Black line is CD4 negative control (CD4 unlabeled CEM-T4); black dotted line is CD4 positive control (CD4 labeled CEM-T4); gray is CEM-T4 transfected with Nef-GFP (control to look at the effect of Nef expression on CD4 surface expression in CEM-T4); blue is CEM-T4 cells co-cultured with CdCl2 treated N5 cells; red is CEM-T4 cells co-cultured with untreated N5 cells; yellow is CEMT4 cells co-cultured with untreated RAW 264.7 cells; green is CEM-T4 cells co-cultured with CdCl2 treated RAW 264.7 cells. (e) Graphical representation of (c-d) plotting the percent of CEM-T4 cells with a Mean Fluorescence Intensity (MFI) below that of positive control cells (CD4 labeled CEM-T4) from 7 independent flow cytometry experiments for all 4 mixes conditions. Co-culture of CdCl2 treated N5 cells with CEM-T4 cells induces a statistically significant decrease of the MFI of CEM-T4 cells (c, d and E- blue). The graph shows the means (± s.e.m), with a P value <0.001 for all 4 conditions (***)

Article Snippet: Aqua-poly mount (Cat # 18606) was purchased from PolySciences, Inc. Rabbit anti-Myo10 (Cat # HPA024223) and rabbit anti-calnexin (Cat # C4731) were purchased from Sigma; Guinea Pig anti-Nef (Cat # APP4963) and PerCP-Cy 5.5 mouse anti-human CD4 (Cat # BDB560650) were purchased from Fisher Scientific; rabbit anti- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sc-25778), goat anti-rabbit IgG-HRP (sc-2004) and goat anti-guinea pig IgG-HRP (sc-2438) were purchased from Santa Cruz Biotechnology, Inc.; and Pacific blue anti-mouse/human CD11b (Cat #101224) was purchased from Biolegends.

Techniques: Flow Cytometry, Staining, Fluorescence, Negative Control, Positive Control, Labeling, Transfection, Expressing, Cell Culture, Co-Culture Assay

Journal: Journal of Cell Communication and Signaling

Article Title: Myosin-X is essential to the intercellular spread of HIV-1 Nef through tunneling nanotubes

doi: 10.1007/s12079-018-0493-z

Figure Lengend Snippet: CEM-T4 were mixed in a 1 to 3 ratio with Raw 264.7 cells and incubated for 24 h with the supernatant from Raw 264.7 (± CdCl2) or the supernatant of N5 (± CdCl2). The mixed population of cells were analyzed by flow cytometry for cell surface CD4 staining. Representative 2-D scatter plots of the CD4 fluorescence intensity (log scale) in CEM-T4 mixed with supernatant from N5 (red) vs supernatant from Raw 264.7 (black) not-treated (a) or treated with CdCl2 (b). Representative CDF (c) and histogram (d) views are plotted. Black dashed line is CD4 negative control; black dotted line is CD4 positive control; gray is Nef expression in CEM-T4; blue is CEM-T4 cells co-cultured with the supernatant of CdCl2 treated N5 cells; red is CEM-T4 cells co-cultured with the supernatant of untreated N5 cells; yellow is CEMT4 cells co-cultured with the supernatant of untreated RAW 264.7 cells; green is CEM-T4 cells co-cultured with the supernatant of CdCl2 treated RAW 264.7 cells. (e) Graphical representation of (c-d) plotting the percent of CEM-T4 cells with a Mean Fluorescence Intensity (MFI) below that of positive control cells (CD4 labeled CEM-T4) from 3 independent flow cytometry experiments for all 4 culture conditions. No statistically significant changes of the MFI of CEM-T4 cells were observed under any conditions

Article Snippet: Aqua-poly mount (Cat # 18606) was purchased from PolySciences, Inc. Rabbit anti-Myo10 (Cat # HPA024223) and rabbit anti-calnexin (Cat # C4731) were purchased from Sigma; Guinea Pig anti-Nef (Cat # APP4963) and PerCP-Cy 5.5 mouse anti-human CD4 (Cat # BDB560650) were purchased from Fisher Scientific; rabbit anti- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sc-25778), goat anti-rabbit IgG-HRP (sc-2004) and goat anti-guinea pig IgG-HRP (sc-2438) were purchased from Santa Cruz Biotechnology, Inc.; and Pacific blue anti-mouse/human CD11b (Cat #101224) was purchased from Biolegends.

Techniques: Incubation, Flow Cytometry, Staining, Fluorescence, Negative Control, Positive Control, Expressing, Cell Culture, Labeling