Background Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte （CTL） numbers,immunophenotype,tumor-specificity,and in vivo residence time,migration,and distribu...Background Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte （CTL） numbers,immunophenotype,tumor-specificity,and in vivo residence time,migration,and distribution.Therefore,tracing in vivo persistence,migration,and distribution of CTLs is important for cancer immunotherapy.Methods Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 （CCK-8） assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester （CFSE）-labeled melanoma antigen-specific cytotoxic T lymphocytes （CFSE-CTLs） for malignant melanoma cells in vitro were compared.Additionally,CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma,and their residence time,migration,and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue （％FI/g） in various tissues and analyzing tumor/non-tumor （T/NT） values.Anti-tumor effects of transferred CTLs and correlation between ％FI/g and D-value of tumor size were analyzed.Results Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity.No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing （P=0.849） or interleukin-2 （P=0.318） and interferon-y （P=0.201）levels.Distribution of CTLs in vivo varied with time.A negative correlation between ％FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed.CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion.CTLs were observed up to three weeks later in the tumor,liver,kidneys,and spleen; this was related to the abundant blood supply or the nature of immune organs.Conclusions CCK-8 assay is a novel method to select optimal CFSE staining concentrations.Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors.CFSE fluorescent markers can trace in vivo CTL persistence,migration,and distribution because of its stability,long half-life,and low toxicity.展开更多
Appressed and non-appressed lamella membranes of Castor bean leaf chloroplasts were separated by non-ionic detergent Triton-X 100.Appressed membranes showed a high oxygen-evolving activity and low chl a/b ratio. Exami...Appressed and non-appressed lamella membranes of Castor bean leaf chloroplasts were separated by non-ionic detergent Triton-X 100.Appressed membranes showed a high oxygen-evolving activity and low chl a/b ratio. Examining with SDS-PTGE and liquid nitrogen temperature fluorescence measurement showed that they contained only PSII and light-harvesting pigment-protein complexes （LHCP）,and there was no detectable amount of PSI. Freeze-fracture electromicroscopic observation confirmed that this part was really an appressed lamella membrane. Through divalent cation Mg++, the thylakoid membranes were induced to unstack and restack.With the addition of Mg++, the fluorescence intensity was changed instantly. We realized that there existed two processes:One was a rapid process which was accomplished within 30 s. The other was a slow process of which the time duration was about 60 min. This dual effects of Mg++ had not been reported before.We had analyzed the change of F685/F730 and discussed the展开更多
文摘Background Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte （CTL） numbers,immunophenotype,tumor-specificity,and in vivo residence time,migration,and distribution.Therefore,tracing in vivo persistence,migration,and distribution of CTLs is important for cancer immunotherapy.Methods Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 （CCK-8） assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester （CFSE）-labeled melanoma antigen-specific cytotoxic T lymphocytes （CFSE-CTLs） for malignant melanoma cells in vitro were compared.Additionally,CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma,and their residence time,migration,and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue （％FI/g） in various tissues and analyzing tumor/non-tumor （T/NT） values.Anti-tumor effects of transferred CTLs and correlation between ％FI/g and D-value of tumor size were analyzed.Results Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity.No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing （P=0.849） or interleukin-2 （P=0.318） and interferon-y （P=0.201）levels.Distribution of CTLs in vivo varied with time.A negative correlation between ％FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed.CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion.CTLs were observed up to three weeks later in the tumor,liver,kidneys,and spleen; this was related to the abundant blood supply or the nature of immune organs.Conclusions CCK-8 assay is a novel method to select optimal CFSE staining concentrations.Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors.CFSE fluorescent markers can trace in vivo CTL persistence,migration,and distribution because of its stability,long half-life,and low toxicity.
文摘Appressed and non-appressed lamella membranes of Castor bean leaf chloroplasts were separated by non-ionic detergent Triton-X 100.Appressed membranes showed a high oxygen-evolving activity and low chl a/b ratio. Examining with SDS-PTGE and liquid nitrogen temperature fluorescence measurement showed that they contained only PSII and light-harvesting pigment-protein complexes （LHCP）,and there was no detectable amount of PSI. Freeze-fracture electromicroscopic observation confirmed that this part was really an appressed lamella membrane. Through divalent cation Mg++, the thylakoid membranes were induced to unstack and restack.With the addition of Mg++, the fluorescence intensity was changed instantly. We realized that there existed two processes:One was a rapid process which was accomplished within 30 s. The other was a slow process of which the time duration was about 60 min. This dual effects of Mg++ had not been reported before.We had analyzed the change of F685/F730 and discussed the
