Journal: RSC Advances

Article Title: CellTrap: an instrument-free microfluidic platform for cell–cell interactions at stochastically generated effector-to-target ratios

doi: 10.1039/d6ra02345b

Figure Lengend Snippet: Characterization of the CellTrap device with 1024 traps. (A) The experimental loading frequencies ( f ) of individual particles, i.e. , green and red, are compared to the theoretical Poisson distributions ( P ) for λ 1 = 0.51 (green) and λ 2 = 0.54 (red). (B) A combinatorial loading distribution of the two particle types was analyzed using a double Poisson distribution, which is compared with the experimental dual loading frequency. (C) Representative bright-field, fluorescence, and overlay images of red and green particles trapped in a channel at different k 1 : k 2 ratios. (D) The experimental loading frequencies ( f ) and the theoretical Poisson distributions ( P ) are plotted for the U87 GFP and NK92 IL2 cells seeded in the CellTrap device with λ 1 = 0.83 (U87 GFP ) and λ 2 = 0.58 (NK92 IL2 ). (E) The experimental dual loading frequency and theoretical double Poisson distributions result in varying E : T or k 1 : k 2 ratios. (F) Representative bright-field, fluorescence, and overlay images of cancer (purple) and immune (white arrows) cells trapped inside a channel at different E : T ratios. (scale bars: 30 µm).

Article Snippet: The human cell lines NK92 IL2 , U87, K562, and LS174T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence

Journal: RSC Advances

Article Title: CellTrap: an instrument-free microfluidic platform for cell–cell interactions at stochastically generated effector-to-target ratios

doi: 10.1039/d6ra02345b

Figure Lengend Snippet: Response of PBMCs and NK92 IL2 against U87 GFP cells. (A) Fluorescence intensity of U87 GFP cells decreases significantly after 4 h of co-incubation with PBMCs at E : T = ≥1 : ≥1. This data is curated from two independent CellTrap devices ( N = 2), where, in total, 97 traps were analyzed ( n = 97). (B) Inside one of the CellTrap devices used in (A), control traps with only one U87 GFP cell per trap, i.e. , E : T = 0 : 1, were analyzed, maintaining a stable fluorescence signal over 14 h ( N = 1, n = 18). (C) Representative time-lapse images of U87 GFP cells interacting with PBMCs at different E : T ratios, along with the control group containing only U87 GFP cells. (D) Fluorescence intensity of U87 GFP cells decreases significantly after 4 h of co-incubation with NK92 IL2 at E : T = 1 : 1. This data is curated from four independent CellTrap devices ( N = 4), where, in total, 213 traps with E : T = 1 : 1 were analyzed ( n = 213). (E) Inside one of the CellTrap devices used in (D), control traps with only one U87 GFP cell per trap, i.e. , E : T = 0 : 1, were analyzed, maintaining a fluorescence signal over 14 h ( N = 1, n = 50). (F) Representative time-lapse images of U87 GFP cells interacting with NK92 IL2 at different E : T ratios, along with the control group containing only U87 GFP cells. (G) Fluorescence intensity of U87 GFP at 0 h and 14 h of co-incubation with NK92 IL2 at E : T = 1 : 1, 1 : 2, 2 : 1 and 2 : 2. The intensity drop is significant across all E : T ratios except 1 : 2. This data is curated from the same CellTrap devices used in (D).

Article Snippet: The human cell lines NK92 IL2 , U87, K562, and LS174T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Incubation, Control

Journal: RSC Advances

Article Title: CellTrap: an instrument-free microfluidic platform for cell–cell interactions at stochastically generated effector-to-target ratios

doi: 10.1039/d6ra02345b

Figure Lengend Snippet: Calcium flux and killing response of immune cells against cancer cells. (A) Calcium flux (normalized intensity) in NK92 IL2 immune cells varies over time in the presence of various cancer cell lines (U87, LS174T, K562). NK92 IL2 cells alone show a flat response (control). Each grey line represents a single immune cell tracked. Representative images show NK92 IL2 cells (green) and cancer cells (U87, LS174T, K562) co-incubated in the CellTrap chip at an E : T ratio of 1 : 1. n = number of traps analyzed. (B) The killing response of NK92 IL2 cells at different E : T ratios (1 : 1, 1 : 2, 2 : 1) against cancer cells (U87, LS174T, K562) is quantified and compared with control groups with E : T ratios of ≥1 : 0 and 0 : ≥1. N = number of CellTrap chips analyzed. n = number of traps analyzed. Representative images show NK92 IL2 cells interacting with cancer cells (U87, LS174T, K562 in blue) with varying E : T ratios at 0 and 14 hours. Red color indicates cell death at 14 hours. Scale bars: 25 µm.

Article Snippet: The human cell lines NK92 IL2 , U87, K562, and LS174T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Control, Incubation

Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Re-engineered segment 8 enables seamless integration of the human IL-2 ORF into the IAV-ΔNS1 vector backbone. ( a ) Schematic representation of alternative splicing for influenza A virus mRNA encoded by wild-type or re-engineered segment 8. Icon descriptions are provided in the figure. Not to scale. ( b ) Representative agarose gel image showing segment 8-specific RT-PCR products amplified from viral RNAs for the indicated viruses. ( c ) Ratios of spliced versus total segment 8 mRNA copy numbers from HEK293T cells infected with viruses carrying wild-type or ΔNS1-IL-2 segment 8 at an MOI of 1, quantified by qRT-PCR, at the indicated time points. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( d ) Western blot images acquired from lysates of HEK293T cells infected with the indicated viruses or mock controls at 24 hours post-infection. A representative image is shown. ( e ) Multi-cycle growth curve analysis for the indicated viruses in MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. IL-2 ELISA ( f ) and IL-2 bioactivity assay ( g ) results for supernatants collected from MDCK-NS1 cells infected with indicated viruses at an MOI of 1 at 24 hpi. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( h ) IL-2 ELISA results for supernatants collected from consecutive blind passages of ∆NS1-IL-2 viruses on MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. bp, base pair; hpi, hours post-infection; IB, immunoblot; kDa, kilodalton; pfu, plaque-forming unit; mL, milliliter; and ng, nanogram.

Article Snippet: The human IL-2 gene, amplified from the commercially available plasmid (RC210013, Origene) and flanked with homologous sequences, was fused to the adjacent segments by nested PCR and cloned into a pDZ vector.

Techniques: Plasmid Preparation, Alternative Splicing, Virus, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Infection, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Novel IAV-ΔNS1 vectors induce notable viral antigen expression and vector-sourced IL-2 secretion in IFN-competent systems. ( a ) Representative images of plaque assays for pre-estimated doses of indicated viruses at 33°C or 37°C on MDCK or MDCK-NS1 cells. Plaques were stained by standard immuno-staining against IAV NP protein. ( b ) Multi-cycle growth curve analysis for the indicated viruses in MDCK cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. ( c and d ) MDCK, A549, and HEK293T cells were infected with the indicated viruses at an MOI of 1. ( c ) At 24 hpi, infection rates were quantified by flow cytometry analysis for IAV NP-expressing cells. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). One-way ANOVA was applied to test for statistical significance. P < 0.05. ( d ) IL-2 levels in culture supernatants and bioactivity of vector-sourced IL-2 were measured at the indicated time points by sandwich ELISA and HEK-Blue CD122/CD132 cells, respectively. Representative data from two independent experiments are depicted as median ( n = 3). ( e and f ) BALB/c mice were intranasally inoculated with the vectors at a dose of 1 × 10 5 pfu/animal or mock controls. ( e ) Animals were monitored for weight loss up to 7 days post-infection ( n = 5). The dotted line indicates the corresponding human endpoint (75% of initial body weight). Data are depicted as mean ± SD. ( f ) IL-2 levels measured by sandwich ELISA in bronchoalveolar lavages (BALs) and serum collected from mice inoculated with the indicated viruses or mock controls at 24 hpi. Data are depicted as mean ± SD. Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. ng, nanogram; mL, milliliter; and hpi, hours post-infection.

Article Snippet: The human IL-2 gene, amplified from the commercially available plasmid (RC210013, Origene) and flanked with homologous sequences, was fused to the adjacent segments by nested PCR and cloned into a pDZ vector.

Techniques: Expressing, Plasmid Preparation, Staining, Immunostaining, Control, Infection, Flow Cytometry, Sandwich ELISA

Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Re-engineered segment 8 enables seamless integration of the human IL-2 ORF into the IAV-ΔNS1 vector backbone. ( a ) Schematic representation of alternative splicing for influenza A virus mRNA encoded by wild-type or re-engineered segment 8. Icon descriptions are provided in the figure. Not to scale. ( b ) Representative agarose gel image showing segment 8-specific RT-PCR products amplified from viral RNAs for the indicated viruses. ( c ) Ratios of spliced versus total segment 8 mRNA copy numbers from HEK293T cells infected with viruses carrying wild-type or ΔNS1-IL-2 segment 8 at an MOI of 1, quantified by qRT-PCR, at the indicated time points. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( d ) Western blot images acquired from lysates of HEK293T cells infected with the indicated viruses or mock controls at 24 hours post-infection. A representative image is shown. ( e ) Multi-cycle growth curve analysis for the indicated viruses in MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. IL-2 ELISA ( f ) and IL-2 bioactivity assay ( g ) results for supernatants collected from MDCK-NS1 cells infected with indicated viruses at an MOI of 1 at 24 hpi. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( h ) IL-2 ELISA results for supernatants collected from consecutive blind passages of ∆NS1-IL-2 viruses on MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. bp, base pair; hpi, hours post-infection; IB, immunoblot; kDa, kilodalton; pfu, plaque-forming unit; mL, milliliter; and ng, nanogram.

Article Snippet: Human IL-2 ( NM_000586 ) and IL-15 ( NM_172174 ) complementary DNAs (cDNAs) were cloned from commercially available plasmids (RC210013 and RC219294, respectively; Origene).

Techniques: Plasmid Preparation, Alternative Splicing, Virus, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Infection, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Novel IAV-ΔNS1 vectors induce notable viral antigen expression and vector-sourced IL-2 secretion in IFN-competent systems. ( a ) Representative images of plaque assays for pre-estimated doses of indicated viruses at 33°C or 37°C on MDCK or MDCK-NS1 cells. Plaques were stained by standard immuno-staining against IAV NP protein. ( b ) Multi-cycle growth curve analysis for the indicated viruses in MDCK cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. ( c and d ) MDCK, A549, and HEK293T cells were infected with the indicated viruses at an MOI of 1. ( c ) At 24 hpi, infection rates were quantified by flow cytometry analysis for IAV NP-expressing cells. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). One-way ANOVA was applied to test for statistical significance. P < 0.05. ( d ) IL-2 levels in culture supernatants and bioactivity of vector-sourced IL-2 were measured at the indicated time points by sandwich ELISA and HEK-Blue CD122/CD132 cells, respectively. Representative data from two independent experiments are depicted as median ( n = 3). ( e and f ) BALB/c mice were intranasally inoculated with the vectors at a dose of 1 × 10 5 pfu/animal or mock controls. ( e ) Animals were monitored for weight loss up to 7 days post-infection ( n = 5). The dotted line indicates the corresponding human endpoint (75% of initial body weight). Data are depicted as mean ± SD. ( f ) IL-2 levels measured by sandwich ELISA in bronchoalveolar lavages (BALs) and serum collected from mice inoculated with the indicated viruses or mock controls at 24 hpi. Data are depicted as mean ± SD. Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. ng, nanogram; mL, milliliter; and hpi, hours post-infection.

Article Snippet: Human IL-2 ( NM_000586 ) and IL-15 ( NM_172174 ) complementary DNAs (cDNAs) were cloned from commercially available plasmids (RC210013 and RC219294, respectively; Origene).

Techniques: Expressing, Plasmid Preparation, Staining, Immunostaining, Control, Infection, Flow Cytometry, Sandwich ELISA

Journal: bioRxiv

Article Title: The Glucose Transporter GLUT3 Controls Regulatory T Cell Function

doi: 10.64898/2026.03.26.714439

Figure Lengend Snippet: (A and B) Relative body weight changes (A) and experimental autoimmune encephalomyelitis (EAE) clinical scores (B) in WT, GLUT1-deficient ( Slc2a1 fl/fl Cd4 Cre ) and GLUT3-deficient ( Slc2a3 fl/fl Cd4 Cre ) mice following immunization with MOG 35-55 peptide emulsified in CFA; means ± SEM of 6-11 mice per cohort. (C) RT-qPCR analysis of Slc2a1 (GLUT1) and Slc2a3 (GLUT3) expression in isolated splenic CD4 + T cells from WT, GLUT1-deficient and GLUT3-deficient mice used in EAE experiments; data are shown as means ± SEM of 4-11 mice. (D and E) Representative flow cytometric analysis of Foxp3 + Treg cells in the central nervous system (CNS) of WT, GLUT1- and GLUT3-deficient mice 20 days after MOG 35-55 immunization (D) , with quantification of Treg cell frequencies and absolute cell numbers (E) ; means ± SEM of 6-11 mice. (F and G) Frequencies of GM-CSF and IL-2-producing CD4 + T cells in the CNS of WT, GLUT1-and GLUT3-deficient mice 20 days after immunization with MOG 35-55 peptide and restimulation with PMA/ionomycin for 5 h (F) , with quantification of total cell numbers of GM-CSF and IL-2 producing CD4 + T cells in the CNS (G) ; means ± SEM of 6-11 mice. Statistical analyses shown in (A, B, C, E, G) were performed using two-way ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001; ns, non-significant.

Article Snippet: FACS-sorted Treg cells were activated on Delta surface 96-well plates (Nunc) with 0.25 ng/mL anti-CD3 (clone 145-2C1) and 1 ng/mL anti-CD28 (clone 37.51; both Bio X Cell), 20 ng/mL recombinant human IL2 and 10 ng/mL murine IL7 for three days at a density of 2×10 6 cells/mL.

Techniques: Quantitative RT-PCR, Expressing, Isolation