Journal: Cell reports

Article Title: Temporal dynamics of immune cell transcriptomics in brain metastasis progression influenced by gut microbiome dysbiosis

doi: 10.1016/j.celrep.2025.115356

Figure Lengend Snippet:

Article Snippet: Following viability stain, cells were blocked in the same manner as described for CITE-seq preparation, using FcR Blocking Reagent (Miltenyi Biotec, 130-092-575) and then stained with one or a combination of the following antibodies: rat anti-mouse CD3e-647 (BioLegend, 155609, KT3.1.1), hamster anti-mouse TCR γ/δ-PE (BioLegend, 118107, GL3), rat anti-mouse CD117(c-kit)-PE-Cy7 (BioLegend, 105813, 2B8), mouse anti-mouse CX3CR1–421 (BioLegend, 149023, SA011F11), rat anti-mouse CD192(CCR2)-510 (BioLegend, 150617, SA203G11), rat anti-mouse CD3-SparkBlue550 (BioLegend, 100259, 17A2), rat anti-mouse CD8-FITC (BioLegend, 100706, 53–6.7), rat anti-mouse CD4–711 (BioLegend, 100549, RM4–5), rat anti-mouse Ly-6C-785 (BioLegend, 128041, HK1.4), rat anti-mouse Ly-6G-650 (BioLegend, 127641, 1A8), rat anti-mouse CD186(CXCR6)-PerCP/Cyanine5.5 (BioLegend, 151120, SA051D1), rat anti-mouse CD11b-605 (BioLegend, 101237, M1/70), rat anti-mouse CD45-PacificBlue (BioLegend, 103125, 30-F11), rat anti-mouse CD45-PerCP/Cyanine5.5 (BioLegend, 103131, 30-F11), rat anti-CD3-FITC (BioLegend, 100203, 17A2), hamster anti-mouse TCR γ/δ-PE-Cy7 (BioLegend, 118123, GL3), rat anti-mouse CD279(PD-1)-421 (BioLegend, 135217, 29F.1A12), rat anti-CD25-PE (BioLegend, 102007, PC61), rat anti-CD4-APC-Cy7 (Tonbo, 25-0042-U025, RM4–5), rat anti-mouse CD8a-710 (Tonbo, 80-0081-U025, 53–6.7), rat anti-mouse CD3-PacificBlue (BioLegend, 100213, 17A2), rat anti-mouse CD4–570 (BioLegend, 100541, RM4–5), rat anti-mouse CD8a-APC-Cy7 (BioLegend, 100713, 53–6.7), hamster anti-mouse TCR γ/δ−711 (BioLegend, 118149, GL3), mouse anti-mouse NK-1.1-SparkRed718 (BioLegend, 156533, S17016D), rat anti-mouse CD25–750 (BioLegend, 102077, PC61), mouse anti-mouse CD159a(NKG2A B6 )-PE (BioLegend, 142803, 16A11), rat anti-mouse CD107a(LAMP-1)-PE-Cy7 (BioLegend, 121619, 1D4B), rat anti-mouse/human CD11b-650 (BioLegend, 101259, M1/70), rat anti-mouse CD8a-647 (BioLegend, 100727, 53–6.7), rat anti-mouse CD279(PD-1)-421 (BioLegend, 135221, 29F.1A12), rat anti-mouse FOXP3–647 (BioLegend, 126407, MF-14).

Techniques: In Vivo, Recombinant, Blocking Assay, Red Blood Cell Lysis, Multiplex Assay, Sequencing, Software, Amplification, Fluorescence

Journal: Cell reports

Article Title: Temporal dynamics of immune cell transcriptomics in brain metastasis progression influenced by gut microbiome dysbiosis

doi: 10.1016/j.celrep.2025.115356

Figure Lengend Snippet:

Article Snippet: Flow: rat anti-CD4-APC-Cy7 (RM4–5) , Tonbo , Cat#25–0042-U025; RRID: N/A.

Techniques: In Vivo, Recombinant, Blocking Assay, Red Blood Cell Lysis, Multiplex Assay, Sequencing, Software, Amplification, Fluorescence

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: Impact of OPLS on RTX- and ADM-mediated T-cell activation when administered subcutaneously in mice. CD4 + T cells in the draining lymph nodes (DLN) of mice at the study terminal were stained for the activation marker CD44 and classical immune checkpoints CLTA-4 and PD-1. Supplementary Figure 3 is the flow cytometry gating strategy. Frequency (%) of (A) CD44 ++ and (B) CTLA-4 + PD-1 + cells out of live CD3 + CD4 + T cells in DLN of mice administered RTX + rHuPH20 in vehicle, 25 mM OPLS, or 100 mM OPLS or (C) in DLN of mice administered ADM in vehicle, 25 mM OPLS, or 50 mM OPLS. Dots represent individual mice and bars are mean ± SD. Statistical significance was determined by Student’s unpaired t-test (one-tailed). *p<0.05, **p<0.01.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: Activation Assay, Staining, Marker, Flow Cytometry, One-tailed Test

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: Co-administration with OPLS promotes differentiation of regulatory T cells in vivo and in vitro . In the ADM study ( Figure 1 ), mouse splenocytes were collected at the terminal endpoint and analyzed for Tr1 phenotype. Supplementary Figure 4 is the flow cytometry gating strategy. (A) Frequency (%) of LAG-3 + CD49b + FoxP3 - CD4 + Tr1 cells in the spleen. Dots represent individual mice and bars are mean ± SD. Statistical significance was determined by Student’s unpaired t-test (one-tailed). *p<0.05. (B, C) Naïve immature SW mouse BMDCs were cultured for 24 h with media, VitD3/Dex, OVA (a model antigen), and OVA + 25 mM or 50 mM OPLS. Splenocytes from SW mice (n=2) subcutaneously immunized with OVA (2 μg/100 μL) once weekly were collected 4 days after the second dose. Isolated CD4 + T cells were co-cultured with treated BMDCs at a ratio of 1:5 BMDC, CD4 + T cell for 72 h. Frequency (%) (B) LAG-3 + CD49 + Tr1 and (C) LAP + of CD4 + FoxP3 - T cells. Error bars are mean ± SD of triplicate wells.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: In Vivo, In Vitro, Flow Cytometry, One-tailed Test, Cell Culture, Isolation

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: OPLS does not impact immunocompetence of NHP and mice. CD-1 mice received daily SC doses of vehicle (n=6), 20 mg/kg CYP (n=3), or OPLS (n=6/group) at 18-450 mM for 28 days. NHP (rhesus macaques) (n=3) received 21 daily SC doses of 54 mM OPLS. (A, B) Frequencies of lymphocyte populations—CD19 + B cells, CD3 + T cells, CD3 + CD4 + T cells, and CD3 + CD8 + T cells—in (A) spleens of mice on day 28 and (B) peripheral blood of NHP on day 0 (baseline), 8, 18, 22, and 35. Supplementary Figure 5 is the gating strategy. (C) Anti-KLH IgM and (D) IgG titers (log 2 ) in plasma of mice on day 28 following a single IV dose of KLH (2 mg) on day 15. (E) Anti-KLH IgM and IgG titers (log 2 ) in plasma of NHP on day 18 and 22, respectively, following a single IM dose of KLH (10 mg) on day 8. Each dot represents an individual animal and all bars are mean ± SD. (F-K) Microscopic images of H&E-stained spleens from mice treated with (F) vehicle or (G) 18 mM, (H) 45 mM, (I) 90 mM, (J) 225 mM, and (K) 450 mM OPLS. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. ****p<0.0001.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: Clinical Proteomics, Staining