vva fitc  (Vector Laboratories)

Journal: Mucosal immunology

Article Title: Dendritic cell-mediated responses to secreted Cryptosporidium effectors promote parasite-specific CD8 + T cell responses

doi: 10.1016/j.mucimm.2024.03.003

Figure Lengend Snippet: The use of a model antigen to generate parasite-specific CD8 + T cell responses. (A) Genetic construct of transgenic M2-OVA Cryptosporidium parasites with the Neo selection marker and the Nluc reporter to monitor parasite burden. (B) HCT-8 cells were infected for 9 hours and then stained for nuclear dye, Hoechst (blue), glycans, Vicia villosa lectin conjugated to FITC (VVL) (green), and HA (red). A white arrow points to the parasite within the cell in addition to the HA staining in the parasite. Uninfected cells are labeled with UI and infected cells are labeled with I. (C) IFN-γ −/− mice received 10 4 OT-I cells and were infected with 10 4 WT Cp or M2-OVA parasites and IEL was harvested at 10 dpi for flow cytometry. Representative flow plots show OT-I cells in the IEL, gated on Singlets, Live, CD45.2 + , CD19 − , NK1.1 − , CD3 + , CD4 − , CD8α + , SIINFEKL:K b+ , CD45.1 + . Summary bar graph showing percentages of OT-I cells from n = 2–4 mice/group from 2–6 experiments with dots representing individual mice. (D) IFN-γ −/− mice were infected with 10 4 M2-OVA and mLN and IEL were harvested at 10 dpi for flow cytometry. Representative flow plots show endogenous SIINFEKL:K b+ cells gated on Singlets, Live, CD19 − , NK1.1 − , B220 − , CD3 + , CD4 − , CD8α + , SIINFEKL:K b+ . Summary bar graph of one representative experiment showing endogenous SIINFEKL:K b+ frequency in mLN and IEL from four individual mice, represented by dots. n = 2–4 mice/group from three experiments. Statistical significance was determined in (C) and (D) by Student’s t test with Welch’s correction * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CD = cluster of differentiation; dpi = days post infection; FITC = fluorescein isothiocyanate; HA = hemagglutinin; HCT = human ileocecal adenocarcinoma cells; IEL = intraepithelial lymphocyte; IFN = interferon; M2 = MEDLE-2 ; mLN = mesenteric lymph node; Neo = neomycin; NK = natural killer; Nluc = nanoluciferase; ; OVA = ovalbumin; ; UI = uninfected; VVL = vicia villosa lectin; WT = wildtype.

Article Snippet: Rat monoclonal anti-HA (clone: 3F10; Sigma) was used as primary antibody and goat anti-rat polyclonal Alexa Fluor 594 (Thermo Fisher) as secondary along with VVL conjugated to FITC (Vector Labs, Newark, CA, USA).

Techniques: Construct, Transgenic Assay, Selection, Marker, Infection, Staining, Labeling, Flow Cytometry

Journal: Mucosal immunology

Article Title: Dendritic cell-mediated responses to secreted Cryptosporidium effectors promote parasite-specific CD8 + T cell responses

doi: 10.1016/j.mucimm.2024.03.003

Figure Lengend Snippet: Secretion of Cryptosporidium antigens is required to induce CD8 + T cell responses. (A) Genetic constructs of transgenic M2-OVA, NS-M2-OVA, and ROP1-OVA Cryptosporidium parasites are shown. (B) HCT-8 cells were infected with M2-OVA or NS-M2-OVA for 9 hours then stained for nuclear dye, Hoechst (blue), glycans, Vicia villosa lectin conjugated to FITC (VVL) (green), and HA (red). A white arrow points to the parasite within the cell in addition to the HA staining in the parasite. Additionally, HCT-8 cells were infected with ROP1-OVA for 2 hours then stained for nuclear dye, Hoechst (blue), glycans, VVL (green), and HA (red). White arrows point to the parasite within the cell as well as the HA staining near the site of infection and around the cell periphery. Uninfected cells are labeled with UI and infected cells are labeled with I. (C) IFN-γ −/− mice received 10 4 OT-I cells and were infected with 10 4 -5 × 10 4 NS-M2-OVA or M2-OVA parasites and mLN, PP, and IEL were harvested at 10 dpi for flow cytometry. Representative flow plots show OT-I cells in IEL gated on Singlets, Live, CD19 − , NK1.1 − , CD90.2 + , CD4 − , CD8α + , CD45.1 + , Va2 + . Summary bar graph of one representative experiment showing percentages of OT-I cells in each tissue from three individual mice, represented by dots. n = 2–3 mice/group for each experiment and has been performed three times. The 5 × 10 4 NS-M2-OVA infection group was only performed once. (D) IFN-γ −/− mice received 10 4 OT-I cells and were infected with 10 4 ROP1-OVA or M2-OVA parasites and mLN, PP, and IEL were harvested at 10 dpi for flow cytometry. Representative flow plots show OT-I cells in IEL gated similarly to (C) Summary bar graph of one representative experiment showing percentages of OT-I cells in each tissue of three individual mice, represented by dots. n = 3 mice/group from three independent experiments. Statistical differences in (C) and (D) were determined based on two-way ANOVA and multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CD = cluster of differentiation; dpi = days post infection; FITC = fluorescein isothiocyanate; HA = hemagglutinin; HCT = human ileocecal adenocarcinoma cell; IEL = intraepithelial lymphocyte; IFN = interferon; M2 = MEDLE-2 ; mLN = mesenteric lymph node; NK = natural killer; ; OVA = ovalbumin; PP = Peyer’s patches; ROP1 = rhoptry protein 1; UI = uninfected; VVL = vicia villosa lectin.

Article Snippet: Rat monoclonal anti-HA (clone: 3F10; Sigma) was used as primary antibody and goat anti-rat polyclonal Alexa Fluor 594 (Thermo Fisher) as secondary along with VVL conjugated to FITC (Vector Labs, Newark, CA, USA).

Techniques: Construct, Transgenic Assay, Infection, Staining, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: Dendritic cell-mediated responses to secreted Cryptosporidium effectors are required for parasite-specific CD8 + T cell responses

doi: 10.1101/2023.08.16.553566

Figure Lengend Snippet: A. Genetic construct of transgenic M2-OVA Cryptosporidium parasites with the neomycin (Neo) selection marker and the nanoluciferase (Nluc) reporter to monitor parasite burden. B. HCT-8 cells were infected for 9 hours then stained for nuclear dye, Hoechst (blue), glycans, Vicia villosa lectin conjugated to FITC (VVL) (green), and HA (red). A white arrow points to the parasite within the cell in addition to the HA staining in the parasite. C. IFN-γ -/- mice received 10 4 OT-I cells and were infected with 10 4 WT Cp or M2-OVA parasites and IEL was harvested at 10 dpi for flow cytometry. Representative flow plots show OT-I cells in the IEL, gated on Singlets, Live, CD45.2 + , CD19 - , NK1.1 - , CD3 + , CD4 - , CD8α + , SIINFEKL:K b+ , CD45.1 + . Summary bar graph showing means of n=2-4 mice/group from 2-6 experiments. D. IFN-γ -/- mice received none or 5×10 4 OT-I then were infected with 10 4 M2-OVA and mLN and IEL were harvested at 10 dpi for flow cytometry. Representative flow plots show SIINFEKL:K b+ cells gated on Singlets, Live, CD19 - , NK1.1 - , B220 - , CD3 + , CD4 - , CD8α + , SIINFEKL:K b+ , CD45.1 + . Summary bar graph of one representative experiment showing means of endogenous SIINFEKL:K b+ and OT-I CD8 + T cell frequency in mLN and IEL. N=2-4 mice/group from 2 experiments. Statistical significance was determined in C by Student’s t test with Welch’s correction * p≤0.05.

Article Snippet: Rat monoclonal anti-HA (clone: 3F10; Sigma) was used as primary antibody and goat anti-rat polyclonal Alexa Fluor 594 (Thermo Fisher) as secondary along with Vicia villosa lectin conjugated to FITC (Vector Labs).

Techniques: Construct, Transgenic Assay, Selection, Marker, Infection, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Dendritic cell-mediated responses to secreted Cryptosporidium effectors are required for parasite-specific CD8 + T cell responses

doi: 10.1101/2023.08.16.553566

Figure Lengend Snippet: A. Genetic constructs of transgenic M2-OVA, NS-M2-OVA, and ROP1-OVA Cryptosporidium parasites are shown. B. HCT-8 cells were infected with M2-OVA or NS-M2-OVA for 9 hours then stained for nuclear dye, Hoechst (blue), glycans, Vicia villosa lectin conjugated to FITC (VVL) (green), and HA (red). A white arrow points to the parasite within the cell in addition to the HA staining in the parasite. Additionally, HCT-8 cells were infected with ROP1-OVA for 2 hours then stained for nuclear dye, Hoechst (blue), glycans, VVL (green), and HA (red). White arrows point to the parasite within the cell as well as the HA staining near the site of infection and around the cell periphery. C. IFN-γ -/- mice received 10 4 OT-I cells and were infected with 10 4 -5×10 4 NS-M2-OVA or M2-OVA parasites and mLN, PP, and IEL were harvested at 10 dpi for flow cytometry. Representative flow plots show OT-I cells in IEL gated on Singlets, Live, CD19 - , NK1.1 - , CD90.2 + , CD4 - , CD8α + , CD45.1 + , Vα2 + . Summary bar graph of one representative experiment showing percentages of OT-I cells in each tissue. N=2-3 mice/group for each experiment and has been performed 3 times. The 5×10 4 NS-M2-OVA infection group was only performed once. D. IFN-γ -/- mice received 10 4 OT-I cells and were infected with 10 4 ROP1-OVA or M2-OVA parasites and mLN, PP, and IEL were harvested at 10 dpi for flow cytometry. Representative flow plots show OT-I cells in IEL gated similarly to C . Summary bar graph of one representative experiment showing percentages of OT-I cells in each tissue. n=3 mice/group from 3 independent experiments. Statistical differences in C and D were determined based on two-way ANOVA and multiple comparisons. * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.

Article Snippet: Rat monoclonal anti-HA (clone: 3F10; Sigma) was used as primary antibody and goat anti-rat polyclonal Alexa Fluor 594 (Thermo Fisher) as secondary along with Vicia villosa lectin conjugated to FITC (Vector Labs).

Techniques: Construct, Transgenic Assay, Infection, Staining, Flow Cytometry