apoptosis is a type of genetically regulated programmed cell death that controls the development of multicellular organisms and tissues by eliminating physiologically redundant , physical damaged , and abnormal cells . studies focusing on the genes and signals regulating apoptosis have played an important role in basic oncology research . radiotherapy and chemotherapy are essential for the treatment of malignant tumors in pre- and post - operative cancer patients . the efficacy of anticancer drugs is measured by their ability to detect cancer cells and selectively promote their apoptosis . the tumor drug sensitivity test ( dst ) is a method to identify the most effective drug to treat tumors based on their sensitivity . the genotype and pathogenesis of tumors vary , and malignant tumors are made up of a polymorphous , heterogeneous , and multi - differentiated cell population . tumors can be resistant to one or more drugs , or exhibit sensitivity towards many drugs . by reducing drug resistance and increasing the efficiency of dsts , multiple methods to detect apoptosis have been developed that detect changes in cell morphology and surface markers associated with apoptosis . this can obscure the result of dsts , affecting the accuracy and validity of the drugs they select . dual acridine orange / ethidium bromide ( ao / eb ) fluorescent staining , visualized under a fluorescent microscope , can be used to identify apoptosis - associated changes of cell membranes during the process of apoptosis . dual ao / eb staining was used to examine apoptosis in a human osteosarcoma cell line treated with the tumor inhibitor kappa - selenocarrageenan . we qualitatively and quantitatively analyzed the effects of kappa - selenocarrageenan using the ao / eb staining method . flow cytometry can be used to measure the fluorescence intensity of intracellular ca(2 ) from bone marrow mesenchymal stem cells ( mscs ) . cell - cycle distribution can be determined by flow cytometric analysis using propidium iodide ( pi ) staining and cellular apoptosis can be evaluated by flow cytometry and terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ) assay . by comparing our technique with flow cytometry analysis technology , we concluded that dual ao / eb staining is a simple and accurate method that can be used in tumor dsts . after obtaining irb approval , a human osteosarcoma cell line ( os-732 cells , purchased from beijing jishuitan hospital , beijing , china ) in the logarithmic growth phase were digested with 0.25% trypsin ( hyclone , logan , ut ) . rpmi1604 culture medium ( hyclone , logan , ut ) containing 10% fetal calf serum ( fcs , hyclone , logan , ut ) was deposited in each well of a 96-well plate ( 100 l / well ) . cells were added to a final concentration of 210/ml , and the plates were incubated . cells were left untreated or treated with 30 , 60 , or 120 g / ml of kappa - selenocarrageenan ( shanghai tiancifu biological engineering co. ltd . , shanghai , china ) . the samples in a 96-well plate were divided into 4 groups , with 24 well samples in each group corresponding to different reagent concentrations . after being cultured for 24 h , 48 h , and when cells had sloughed off , suspensions ( 25 l ) were transferred to glass slides . dual fluorescent staining solution ( 1 l ) containing 100 g / ml ao and 100 g / ml eb ( ao / eb , sigma , st . louis , mo ) was added to each suspension and then covered with a coverslip . the morphology of apoptotic cells was examined and 500 cells were counted within 20 min using a fluorescent microscope ( olympus , japan ) . dual acridine orange / ethidium bromide ( ao / eb ) staining method was repeated 3 times at least . cells were trypsinized ( 0.25% trypsin ) 24 h after kappa - selenocarrageenan was applied to the sample . single - cell suspensions ( 210 cells ) were extracted and washed using phosphate - buffered saline . 0.1% triton x-100 ph 7.8 ) was added to the samples and was incubated at room temperature for 45 min . next , the cells were digested with 50 g / ml rnase for 10 min . cells were stained with pi ( 50 g / ml ) for 30 min ( pi , sigma , st . the samples were analyzed using a flow cytometer ( bd , franklin lakes , nj ) . the cells with dna content less than that of cells at the g1 phase were identified as apoptotic cells . also , flow cytometry analysis was repeated at least 3 times . all statistical analyses were performed using the spss 20.0 software ( spss inc . , chicago , il , usa ) for windows . after obtaining irb approval , a human osteosarcoma cell line ( os-732 cells , purchased from beijing jishuitan hospital , beijing , china ) in the logarithmic growth phase were digested with 0.25% trypsin ( hyclone , logan , ut ) . rpmi1604 culture medium ( hyclone , logan , ut ) containing 10% fetal calf serum ( fcs , hyclone , logan , ut ) was deposited in each well of a 96-well plate ( 100 l / well ) . cells were added to a final concentration of 210/ml , and the plates were incubated . cells were left untreated or treated with 30 , 60 , or 120 g / ml of kappa - selenocarrageenan ( shanghai tiancifu biological engineering co. ltd . , shanghai , china ) . the samples in a 96-well plate were divided into 4 groups , with 24 well samples in each group corresponding to different reagent concentrations . after being cultured for 24 h , 48 h , and when cells had sloughed off , suspensions ( 25 l ) were transferred to glass slides . dual fluorescent staining solution ( 1 l ) containing 100 g / ml ao and 100 g / ml eb ( ao / eb , sigma , st . louis , mo ) was added to each suspension and then covered with a coverslip . the morphology of apoptotic cells was examined and 500 cells were counted within 20 min using a fluorescent microscope ( olympus , japan ) . dual acridine orange / ethidium bromide ( ao / eb ) staining method was repeated 3 times at least . cells were trypsinized ( 0.25% trypsin ) 24 h after kappa - selenocarrageenan was applied to the sample . single - cell suspensions ( 210 cells ) were extracted and washed using phosphate - buffered saline . 0.1% triton x-100 ph 7.8 ) was added to the samples and was incubated at room temperature for 45 min . next , the cells were digested with 50 g / ml rnase for 10 min . cells were stained with pi ( 50 g / ml ) for 30 min ( pi , sigma , st . the samples were analyzed using a flow cytometer ( bd , franklin lakes , nj ) . the cells with dna content less than that of cells at the g1 phase were identified as apoptotic cells . also , flow cytometry analysis was repeated at least 3 times . all statistical analyses were performed using the spss 20.0 software ( spss inc . , chicago , il , usa ) for windows . osteosarcoma cells were labeled by ao / eb 24 h after kappa - selenocarrageenan was applied . early - stage apoptotic cells , marked by crescent - shaped or granular yellow - green ao nuclear staining , were detected in the experimental group ( figure 1b ) . staining was localized asymmetrically within the cells . with increasing concentrations and treatment lengths , the number of early - stage apoptotic cells increased . late - stage apoptotic cells , with concentrated and asymmetrically localized orange nuclear eb staining , were also detected ( figure 1c ) . necrotic cells increased in volume and showed uneven orange - red fluorescence at their periphery . the hypodiploid peak appearing to the left of the g1 peak in the cell cycle histogram formed as a result of apoptosis . no distinctive apoptotic peak was observed for the negative control group cultured for 24 h ( figure 2a ) . the apoptotic peak to the left of the g1 peak immediately appeared 24 h after 30 g / ml of kappa - selenocarrageenan was applied to the osteosarcoma cells ( figure 2b ) . a student s t - test showed no significant difference between the 2 methods ( table 1 ) . osteosarcoma cells were labeled by ao / eb 24 h after kappa - selenocarrageenan was applied . early - stage apoptotic cells , marked by crescent - shaped or granular yellow - green ao nuclear staining , were detected in the experimental group ( figure 1b ) . staining was localized asymmetrically within the cells . with increasing concentrations and treatment lengths , the number of early - stage apoptotic cells increased . late - stage apoptotic cells , with concentrated and asymmetrically localized orange nuclear eb staining , were also detected ( figure 1c ) . necrotic cells increased in volume and showed uneven orange - red fluorescence at their periphery . the hypodiploid peak appearing to the left of the g1 peak in the cell cycle histogram formed as a result of apoptosis . no distinctive apoptotic peak was observed for the negative control group cultured for 24 h ( figure 2a ) . the apoptotic peak to the left of the g1 peak immediately appeared 24 h after 30 g / ml of kappa - selenocarrageenan was applied to the osteosarcoma cells ( figure 2b ) . a student s t - test showed no significant difference between the 2 methods ( table 1 ) . greater than 80% of patients have systemic metastatic cancer when they enter the hospital , with the lungs being the most common site of osteosarcoma metastasis . the long - term survival rate of the patients receiving surgical interventions is only 2030% . over the past 30 years , rosen has been a strong proponent and has advocated for high - dose systemic chemotherapy , namely neoadjuvant chemotherapy , prior to surgical intervention , which has been a milestone in the history of osteosarcoma treatment . this approach has greatly improved the 5-year survival rate of patients with osteosarcoma ; however , nearly half of patients die from pulmonary metastasis because cancers become resistant to chemotherapy drugs within 23 years of treatment . because tumors are composed of a polymorphous , heterogeneous , and multi - differentiated cell population , their sensitivity to various chemotherapeutic drugs differs depending on the individual . the action and metabolism of drugs in vivo is a complex process , and chemotherapy is time - consuming and cost - ineffective . individualized treatment strategies have been proposed . in individualized treatment , dsts are carried out in vitro to identify and select the most effective chemotherapeutics , and to determine its effective dose . therefore , individualized medication could improve the targeting and efficacy of chemotherapeutic agents and reduce adverse reactions and drug resistance multiple methods , such as pi , in situ nick translation , terminal deoxynucleotidyl , acidic denaturation , and thermal denaturation assays , have been developed to detect apoptosis by monitoring changes in cell morphology and surface markers . these methods involve multi - step procedures , including diversion , washing , and transfer of samples , using both time and materials . mtt assays have been used as dsts in vitro , but this method has some weaknesses . for example , this assay can not distinguish between apoptotic and necrotic cells , and therefore can not detect the toxic effects of drugs on the body . while tumor cells undergo apoptosis in the presence of anticancer drugs , normal cells become necrotic if the drug is toxic . therefore , detection of tumor cell apoptosis is more valuable than generally assessing tumor cell viability . in addition , it allows for the distinction between normal cells , early and late apoptotic cells , and necrotic cells . therefore , ao / eb staining is a qualitative and quantitative method to detect apoptosis . we speculate that ao penetrated normal and early apoptotic cells with intact membranes , fluorescing green when bound to dna . eb only entered cells with damaged membranes , such as late apoptotic and dead cells , emitting orange - red fluorescence when bound to concentrated dna fragments or apoptotic bodies . furthermore , dual ao / eb staining is able to detect mild dna injuries . therefore , to distinguish normal , early apoptotic , late apoptotic cells , and dead cells , nuclear morphology must be assessed . fluorescent staining using ao alone has been used in the past ; however , detection of cell apoptosis using ao / eb is a relatively new approach , and few papers have reported its use . in comparison to ao staining , the ao / eb method improves the detection of apoptosis and can distinguish between late apoptotic and dead cells . flow cytometry is a classical method to detect cell apoptosis with high sensitivity and can be used for simultaneous cell - cycle analysis . however , a general laboratory may not be equipped with a flow cytometer , and they are costly to run . no significant difference was observed between the ability of flow cytometry and ao / eb staining to detect apoptosis . therefore , fluorescent staining can feasibly be applied to evaluate apoptosis in dsts of osteosarcomas or other malignant tumors .