Basic Information
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mRNA expression analysis
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Strain specific analysis of C5AR1 gene expression in wild-type C57BL/6 mice and homozygous B-hC5AR1 mice by RT-PCR. Mouse C5ar1 mRNA was detectable in lung tissue of wild-type C57BL/6 mice (+/+). Human C5AR1 mRNA was detectable only in homozygous B-hC5AR1 mice (H/H) but not in wild-type mice.
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Protein expression analysis
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Protein expression analysis (spleen）
Species-specific C5AR1 protein expression analysis in wild-type and humanized B-hC5AR1 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hC5AR1 (H/H) mice, and analyzed by flow cytometry using species-specific anti-C5AR1 antibodies. Murine C5AR1 protein was detected in wild-type mice, while human C5AR1 protein was exclusively detected in B-hC5AR1 mice.
Protein expression analysis (bone marrow）
Species-specific C5AR1 protein expression analysis in wild-type and humanized B-hC5AR1 mice. Bone marrow was isolated from wild-type C57BL/6 (+/+) and homozygous B-hC5AR1 (H/H) mice, and analyzed by flow cytometry using species-specific anti-C5AR1 antibodies. Murine C5AR1 protein was detected in wild-type mice, while human C5AR1 protein was detected in B-hC5AR1 mice.
Protein expression analysis (MC38 cell line & MC38 intratumoral)
Strain specific C5AR1 expression analysis in MC38 cell line and MC38 intratumora by flow cytometry.
MC38 cells were subcutaneously inoculated into C57BL/6 mice, and tumors were removed and prepare tumor cells when tumor volume reached appropriate size. MC38 cells and MC38 intratumora cells were collected and analyzed by flow cytometry with species-specific anti-C5AR1 antibody. Mouse C5AR1 was detectable in intratumora CD45+ cells but not in MC38 cells and intratumora CD45- cells.
Strain specific C5a expression analysis in MC38 cell culture supernatant and MC38 tumor tissue grinding solution by flow ELISA. MC38 cells were subcutaneously inoculated into C57BL/6 mice, tumors were removed and prepare when tumor volume reached appropriate size. MC38 cell culture supernatant and MC38 tumor tissue grinding solution were collected and analyzed by ELISA with mice complement C5a ELISA kit. Mouse C5A was detectable in tumor tissues suspension but not in MC38 cell culture supernatant.
Protein expression analysis (Pan02 cell line & Pan02 intratumoral)
Strain specific C5AR1 expression analysis in Pan02 intratumora by flow cytometry.
Pan02 cells were subcutaneously inoculated into C57BL/6 mice, and tumors were removed and prepare tumor cells when tumor volume reached appropriate size. Pan02 intratumora cells were collected and analyzed by flow cytometry with species-specific anti-C5AR1 antibody. Mouse C5AR1 was detectable in intratumora mCD11b+mGr-1+ cells and mCD11b+mGr-1+mLy-6G+ cells.
Strain specific C5a expression analysis in Pan02 cells, MC38 cells culture supernatant and Pan02, MC38 tumor tissue grinding solution by ELISA.
Pan02 and MC38 cells were subcutaneously inoculated into C57BL/6 mice, tumors were removed and prepare when tumor volume reached appropriate size. Pan02 cells, MC38 cells culture supernatant and Pan02, MC38 tumor tissue grinding solution were collected and analyzed by ELISA with mice complement C5a ELISA kit. Mouse C5a was detectable in tumor tissus suspension but not in cell culture supernatant.
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Immune cell profile
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Analysis of leukocytes cell subpopulation in spleen
Analysis of leukocytes subpopulation in spleen
Splenocytes were isolated from C57BL/6 and B-hC5AR1 mice (n=3). The proportion of leukocytes subpopulation was tested by flow cytometry. As a result, the expression profile of leukocytes subpopulation in homozygous and B-hC5AR1 mice is similar to that in the C57BL/6 mice, indicating that differentiation of T, B, NK, Monocyte, DC and macrophage cells are not affected by the humanization of B-hC5AR1 mice.
Analysis of T cell subpopulation in spleen
Analysis of T cell subpopulation in spleen
The lymphocytes were isolated from spleen in C57BL/6 and B-hC5AR1 mice (n=3). The proportion of T cells subpopulation was tested by flow cytometry.
Analysis of leukocytes cell subpopulation in lymph node
Analysis of leukocytes subpopulation in lymph node
Thymocytes were isolated from C57BL/6 and B-hC5AR1 mice (n=3). The proportion of leukocytes subpopulation was tested by flow cytometry. As a result, the expression profile of leukocytes subpopulation in homozygous and B-hC5AR1 mice is similar to that in the C57BL/6 mice, indicating that differentiation of T, B, NK, Monocyte, DC and macrophage cells are not affected by the humanization of B-hC5AR1 mice.
Analysis of T cell subpopulation in lymph node
Analysis of T cell subpopulation in lymph node
The lymphocytes were isolated from lymph node in C57BL/6 and B-hC5AR1 mice (n=3). The proportion of T cells subpopulation was tested by flow cytometry.
Analysis of leukocytes cell subpopulation in blood
Analysis of leukocytes subpopulation in Blood
Blood were isolated from C57BL/6 (n=2) and B-hC5AR1 mice (n=3). The proportion of leukocytes subpopulation was tested by flow cytometry. As a result, the expression profile of leukocytes subpopulation in homozygous B-hC5AR1 mice is similar to that in the C57BL/6 mice, indicating that differentiation of T, B, NK, Monocyte, DC and macrophage cells are not affected by the humanization of B-hC5AR1 mice.
Analysis of T cell subpopulation in blood
Analysis of T cell subpopulation in Blood
The lymphocytes were isolated from Blood in C57BL/6(n=3) and B-hC5AR1 mice (n=3). The proportion of T cells subpopulation was tested by flow cytometry.
