foxp3 transcription factor staining buffer  (R&D Systems)

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Single-shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immunity in mice.

doi: 10.1016/j.ymthe.2024.05.003

Figure Lengend Snippet: Figure 3. RBD-specific TFH and B cell subsets induced upon single dose immunization with Clec9A-RBD (A) Experimental design schematic to characterize the antigen-specific humoral response elicited in 5- to 6-week-old BALB/c mice (n = 5 per group) s.c. immunized once with 2 mg Clec9A-RBD adjuvanted with 50 mg poly I:C. (B and C) Percentage of spleen RBD-specific (B) GC B cells (B220+ IgD GL-7+ CD95+ RBD++) and (C) MBC (B220+ IgD CD27+ RBD++) measured at 7 days and 1, 6, 9, and 12 months (circle = 6 months, triangle = 9 months, diamond = 12 months) after immunization were determined by flow cytometry. (D) Frequency of bone marrow ASC reactive to ancestral and variant SARS-CoV-2 RBD measured at 1, 6, 9, and 12 months (circle = 6 months, triangle = 9 months, diamond = 12 months), and 21 months after immunization were determined by B cell ELISPOT. Bone marrow cells from naive mice were assessed against ancestral SARS- CoV-2 RBD as a negative control. (E) Percentage of Activation-induced markers positive (AIM+) TFH cells CD3+ CD4+ CXCR5+ PD-1+ CD25+ CD69+) in response to re-stimulation with ancestral and variant SARS-CoV-2 RBD peptides in spleens at 7 days, and 6, 9, and 12 months (circle = 6 months, triangle = 9 months, diamond = 12 months) after immunization were determined by flow cytometry. Splenocytes from naive mice were restimulated with ancestral SARS-CoV-2 RBD peptides as a negative control. (B–E) Symbols represent individual animals and bars represent the mean. Statistical analysis: (B and C) Non-parametric two-tailed Mann-Whitney test or (D and E) Kruskal-Wallis test with Dunnett’s correction for multiple comparisons. *p < 0.05 or **p < 0.01.

Article Snippet: After 30 min incubation at RT, cells were washed twice with 1 Flow Cytometry Perm Buffer prepared as per the manufacturer’s instructions (TNB-0607-KIT; Tonbo Biosciences) before staining with intracellular marker antibodies for 1 h at 4 C in the dark (Table S2).

Techniques: Cytometry, Variant Assay, Enzyme-linked Immunospot, Negative Control, Activation Assay, Two Tailed Test, MANN-WHITNEY

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Single-shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immunity in mice.

doi: 10.1016/j.ymthe.2024.05.003

Figure Lengend Snippet: Figure 4. RBD-specific cellular responses induced upon single dose immunization with Clec9A-RBD (A) Experimental design schematic to characterize the antigen-specific cellular response elicited in 5- to 6-week- old BALB/c mice (n = 5 per group) s.c. immunized once with 2 mg Clec9A-RBD adjuvanted with 50 mg poly I:C. (B) Frequency of total IFN-g+ and IL-5+ splenocytes upon re-stimulation with ancestral and variant SARS- CoV-2 RBD peptides at 7 days after immunization were determined by IFN-g/IL-5 FluoroSPOT. (C) Frequency of splenocytes producing IFN-g, IL-2, and/or TNF-a upon re-stimulation with ancestral and variant SARS-CoV-2 RBD peptides at 7 days after immunization was determined by IFN-g/IL-2/TNF-a FluoroSPOT (B, C). (D) Percentage of spleen CD4+ and CD8+ T cells expressing IFN-g, IL-2, and/or TNF-a upon re-stimulation with ancestral and variant SARS-CoV-2 RBD peptides at 7 days after immunization was determined by flow cytometry. (B–D) Splenocytes from naive mice were restimulated with ancestral SARS-CoV-2 RBD peptides as a negative control. Bars represent the mean ± SD. Statistical analysis: (B–D) Non-parametric two-tailed Kruskal-Wallis test with Dunnett’s correction for multiple comparisons. *p < 0.05.

Article Snippet: After 30 min incubation at RT, cells were washed twice with 1 Flow Cytometry Perm Buffer prepared as per the manufacturer’s instructions (TNB-0607-KIT; Tonbo Biosciences) before staining with intracellular marker antibodies for 1 h at 4 C in the dark (Table S2).

Techniques: Variant Assay, Expressing, Cytometry, Negative Control, Two Tailed Test

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Single-shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immunity in mice.

doi: 10.1016/j.ymthe.2024.05.003

Figure Lengend Snippet: Figure 5. Induction of RBD-specific lung mucosal immune responses upon single dose systemic immunization with Clec9A-RBD (A) Experimental design schematic to characterize the antigen-specific immune response elicited in lung tissues of 5- to 6-week-old BALB/c mice (n = 5 per group) s.c. immunized once with 2 mg Clec9A-RBD adjuvanted with 50 mg poly I:C. (B) Presence of Clec9A-RBD construct in lung homogenate at 4 days after immunization was detected by ELISA. (C) Percentage of RBD-specific GC B cells in perfused lung at 7 days after immunization was determined by flow cytometry. (D) SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a titers in BAL samples at 2 weeks after immunization were determined by ELISA. The dashed line represents the limit of detection (1 log10). The ratio of anti-RBD IgG2a:IgG1 was also calculated. (E and F) Neutralizing and (F) ADCC activity of immune sera and BAL samples against ancestral SARS-CoV-2 at 2 weeks after immunization were determined by c-Pass sVNT and ADCC Reporter Bioassay, respectively. The dashed line represents the cut-off value above which samples are considered positive for neutralizing activity (30%) and baseline ADCC fold induction (1.0), respectively. (G) Percentage of CD4+ and CD8+ T cells expressing IFN-g, IL-2, and/or TNF-a in perfused lung upon re-stimulation with ancestral SARS-CoV-2 RBD peptides at 7 days after immunization were determined by flow cytometry. (B–F) Symbols represent individual animals and bars represent (B, C, and E–G) mean ± (G) SD and (D) geometric mean. Statistical analysis: (B–G) Non-parametric two-tailed Mann-Whitney test. *p < 0.05.

Article Snippet: After 30 min incubation at RT, cells were washed twice with 1 Flow Cytometry Perm Buffer prepared as per the manufacturer’s instructions (TNB-0607-KIT; Tonbo Biosciences) before staining with intracellular marker antibodies for 1 h at 4 C in the dark (Table S2).

Techniques: Construct, Enzyme-linked Immunosorbent Assay, Cytometry, Activity Assay, Bioassay, Expressing, Two Tailed Test, MANN-WHITNEY