Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet: Serum levels of total and HDM-specific immunoglobulins show a time-of-day response and sex-based differences to chronic HDM exposure Total IgE, Total IgG, HDM-specific IgE, HDM-specific IgG, HDM-specific IgG1, HDM-specific IgG2b, HDM-specific IgM, and HDM-specific IgA in the serum of chronic PBS- and HDM-exposed females and males at ZT0 and ZT12 were determined by ELISA. Data were expressed as ng/ml and mg/ml for total IgE and total IgG, respectively. HDM-specific immunoglobulins (IgE, IgG, IgG1, IgG2b, IgM, and IgA) in the serum were determined by commercially available ELISA kits (Chondrex, Inc.). Unable to detect HDM-specific IgG2a and IgG3 levels in PBS- and HDM-exposed mice. Data for the HDM-specific immunoglobulins were expressed as absorbance at 450 nm or μg/ml. Data are shown as mean ± SEM, Two-way ANOVA followed by Tukey’s multiple comparison test (n = 5/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to respective control (PBS) at ZT0 or ZT12; # p < 0.05, compared to HDM at ZT0 vs. ZT12; # # p < 0.01, compared to HDM at ZT0 vs. ZT12. Summary statistics for interaction between Treatment x Sex x Time were analyzed using generalized linear modeling using R (see Table S2 ).

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet:

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: PLoS Pathogens

Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

doi: 10.1371/journal.ppat.1006851

Figure Lengend Snippet: (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

Article Snippet: Then, 300 μl of the cleared lysates from cells transfected with pCAGGS-GST, pCAGGS-GST-PLSCR1, or pCAGGS-GST-NP was mixed with 40 μl of Glutathione Sepharose 4 Fast Flow (GE Healthcare, Pittsburgh, PA) and rocked for 1 h at 4°C.

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Staining, RNA Binding Assay, Infection, Magnetic Beads

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Immunological synapse formation as a key mechanism in T cell-dependent bispecific antibody-mediated immune activation and cytotoxicity

doi: 10.1007/s00262-025-04036-w

Figure Lengend Snippet: Cytotoxic activity and inflammatory cytokine release in co-culture systems. a–c After co-culturing WT or KO, T cells, and TDB, the color reaction with WST-8 reagent in an hour was measured. The co-culture times were 24 h, 48 h, and 72 h of co-culture, respectively. Red: WT + T cell + TDB, blue: KO + T cell + TDB. Error bars represent the mean ± SD ( n = 6), ** p < 0.005, *** p < 0.0005, n.s. not significant. d Measurement of IFN-γ release by ELISA after 24 h of co-culture time. Co-culture supernatants were obtained and used for assay using the ELISA Kit. Red: WT + T cell + TDB, blue: KO + T cell + TDB, gray: negative control (T cell + TDB). Red letters are the results of the WT and N/C statistical tests, and blue letters are the results of the KO and N/C statistical tests. Error bars represent the mean ± SD ( n = 3), *** p < 0.0005, n.s. not significant. e IFN-γ release at TDB: 10 ng/mL for each co-culture time. Error bars represent the mean ± SD ( n ≧ 3), *** p < 0.0005, n.s. not significant. f Cancer cell death when the IFN-γ release measured in e is added to the standard. From left to right, IFN-γ release at 6 h, 24 h, 48 h, and 72 h was added. Error bars represent the mean ± SD ( n ≧ 3), *** p < 0.0005, n.s. not significant. g-i TNF-α version of the same experiment as d – f

Article Snippet: T cells (E:T = 5:1, 1.0 × 10 6 cells/50 μL/well) were added and co-cultured. hEx3 was added at a final concentration of 0.1–100 ng/mL in 100 μL of medium and cultured for 24 h. The culture supernatant was then collected and assayed using human IFN-gamma ELISA Kit, human TNF-alpha ELISA Kit, human CXCL10 ELISA Kit (Proteintech, Rosemont, IL), and human HMGB1 ELISA Kit (Chondrex, Woodinville, WA) according to the protocols provided.

Techniques: Activity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: PLoS ONE

Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

doi: 10.1371/journal.pone.0006209

Figure Lengend Snippet: Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).

Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG 3 anti- S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

Techniques: Blocking Assay, Incubation, Labeling

Journal: PLoS ONE

Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

doi: 10.1371/journal.pone.0006209

Figure Lengend Snippet: Adherent GM-MФs were incubated with unopsonized GFP- S. aureus RN6390. External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. Collapsed confocal stack images were acquired by scanning cytometry. (A) CellTracker Blue and Hoechst channel (cells). (B) GFP channel (all bacteria). (C) Texas Red channel (external bacteria). (D) Composite image. (Original magnification, 200X).

Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG 3 anti- S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

Techniques: Incubation, Labeling, Cytometry