Application of method for transfecting tumor cells by GFP in detecting phagocytic function of tumor-associated macrophages
A technology for macrophages and tumor cells, which is applied in the field of detection of phagocytosis of tumor-associated macrophages. It can solve the problems of not being able to represent the functions of macrophages, poor stability of dyes, short-term in vitro culture, etc., and achieve low cost and stability. Consistent and reliable results
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Embodiment 1
[0065] 1) Human ovarian cancer cell line SKOV3 was cultured, stably transfected with GFP protein, and GFP overexpressed tumor cell line was obtained by puromycin selection for 7 days, and the transfection efficiency was identified by immunofluorescence microscopy and flow cytometry. (GFP plasmid: hU6-MCS-CMV-GFP-SV40-Puromycin)
[0066] 2) Tumor-bearing experiments in mice, BALB / C-nude nude mice were selected to form tumors, 5×10 6 Cells / peritoneal or subcutaneous injection, tumor volume up to 50-100mm 3 Afterwards, follow-up antitumor therapy was carried out. This experiment was divided into control group and PD-1 inhibitor group, 10mg / kg, twice a week, for 4 weeks.
[0067] 3) Separate the mouse tumor tissue after the treatment is completed, and cut the tumor tissue to 1mm 3 Volume, tumor tissue cells were isolated by collagenase digestion. In this experiment, collagenase IV (1.5mg / ml) + hyaluronidase (0.75mg / ml) + DNase (1.1mg / ml) + neutral protease (5U / ml) method was u...
Embodiment 2
[0073] 1) Human ovarian cancer cell line SKOV3 was cultured, stably transfected with GFP protein, and GFP overexpressed tumor cell line was obtained by puromycin selection for 7 days, and the transfection efficiency was identified by immunofluorescence microscopy and flow cytometry.
[0074] 2) Tumor-bearing experiments in mice, BALB / C-nude nude mice were selected to form tumors, 5×10 6 Cells / peritoneal or subcutaneous injection, tumor volume up to 50-100mm 3 Afterwards, follow-up antitumor therapy was carried out. This experiment was divided into control group and CSF1R inhibitor group, 10mg / kg, twice a week, for 4 weeks.
[0075] 3) Separate the mouse tumor tissue after the treatment is completed, and cut the tumor tissue to 1mm 3 Volume, tumor tissue cells were isolated by collagenase digestion. In this experiment, collagenase IV (1.5mg / ml) + hyaluronidase (0.75mg / ml) + DNase (1.1mg / ml) + neutral protease (5U / ml) method was used.
[0076] 4) The phagocytosis of macropha...
Embodiment 3
[0078] 1) Human ovarian cancer cell line SKOV3 was cultured, stably transfected with GFP protein, and GFP overexpressed tumor cell line was obtained by puromycin selection for 7 days, and the transfection efficiency was identified by immunofluorescence microscopy and flow cytometry.
[0079] 2) Tumor-bearing experiments in mice, BALB / C-nude nude mice were selected to form tumors, 5×10 6 Cells / peritoneal or subcutaneous injection, tumor volume up to 50-100mm 3 Afterwards, follow-up antitumor therapy was carried out. This experiment was divided into control group and CSF1R inhibitor group, 25mg / kg, twice a week, for 4 weeks.
[0080] 3) Separate the mouse tumor tissue after the treatment is completed, and cut the tumor tissue to 1mm 3 Volume, tumor tissue cells were isolated by collagenase digestion. In this experiment, collagenase IV (1.5mg / ml) + hyaluronidase (0.75mg / ml) + DNase (1.1mg / ml) + neutral protease (5U / ml) method was used.
[0081] 4) The phagocytosis of macropha...
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