A negative enrichment method for circulating tumor cells
A negative enrichment and tumor cell technology, applied in the field of molecular biology, can solve the problems of low recovery rate of circulating tumor cells, difficulty in selection of biomarkers, inability to obtain active ones, etc., to achieve shortened enrichment time and high sensitivity , the effect of reducing dosage
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Embodiment 1
[0062] A circulating tumor cell negative enrichment method, comprising the following steps:
[0063] S1: Add density gradient separation medium to the lymphocyte separation tube, and centrifuge instantaneously so that the density gradient separation medium is completely centrifuged to the bottom of the tube; add 3 mL of blood samples from tumor patients to the lymphocyte separation tube filled with density gradient separation 3mL of washing buffer, for the first centrifugation, the condition of the first centrifugation is 25°C, 800g / 15min;
[0064] S2: Transfer the upper layer solution in the baffle plate of the above-mentioned lymphocyte separation tube after the first centrifugation to the first centrifuge tube, and perform the second centrifugation. The conditions of the second centrifugation are 25°C, 300g / 10min, suction Remove the supernatant, add 5mL of erythrocyte lysate, let stand for 5min;
[0065] S3: Centrifuge the first centrifuge tube after standing still for the...
Embodiment 2
[0070] Please refer to figure 1 , mixed a certain number of SiHa cells in the peripheral blood of healthy people to simulate the blood samples of patients with cervical cancer containing circulating tumor cells, and evaluated the enrichment efficiency of circulating tumor cell negative enrichment method established, the specific monitoring steps are as follows :
[0071] 1. Pre-label the cultured and digested SiHa cells with Celltracker Green for green fluorescence, and dilute in proportion to obtain 2×10 4 The cell suspension was accurately counted under a fluorescent microscope and adjusted to 100 cells / 5uL;
[0072] 2. Mix the counted cell suspension into 3 mL of peripheral blood to prepare peripheral blood samples containing 100 SiHa cells / 3 mL;
[0073] 3. For peripheral blood samples containing 100 SiHa cells / 3mL, carry out negative enrichment of circulating tumor cells according to the following two schemes respectively;
[0074] A. Traditional density gradient centr...
Embodiment 3
[0078] Please refer to figure 2 and image 3 A certain amount of ovarian cancer SKOV-3, cervical cancer SiHa cells, and breast cancer SKBR3 cells were mixed into the peripheral blood of healthy people to evaluate whether the negative enrichment method of circulating tumor cells involved in this protocol is applicable to circulating tumor cells of different tumor types Detection, the specific detection steps are as follows:
[0079] 1. In this example, 100 ovarian cancer SKOV-3, cervical cancer SiHa cells, and breast cancer SKBR3 cells were mixed into the peripheral blood of healthy people to simulate blood samples containing circulating tumor cells from different tumor patients, cultured, digested and harvested Cells of different tumor types were pre-labeled with Celltracker Green, diluted in proportion to obtain cell suspensions of different concentrations, and counted accurately under a fluorescent microscope.
[0080] 2. The counted cell suspension was mixed into 3 mL of...
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