How can cell pollution be saved?
The experimental method
1) antibiotic method:
Bacterial culture: cultured cells with no antibiotics fully cultured cells 3 d, collected the centrifugation after centrifugation and inoculated the sediment into the blood AGAR medium, 35e culture.
Adopted by the clinical commonly used antibiotics, including 15 kinds of antibiotics, the optimal bacteriostatic antibiotic concentration screening: choose the sensitive antibiotics according to the results of the bacteria medicine sensitive experiment, each antibiotics from the minimal inhibitory concentrations (MIC, respectively is resistance to the highest concentration of six concentration gradient.
The lsc-1 cells were divided into 1 * 104 / foramen 96-well plate, and the cell wall was adherent to the complete medium containing the sensitive antibiotic. After 24 h, it was changed without antibiotic culture 1 d, so repeated 3 times, 7 d consecutively.
2) antibiotic combined macrophage phagocytosis:
The treated cells were treated with macrophages in mice.
Macrophage preparation: will the sterilization of glass powder with 0. 9% sodium chloride injection mixed injected into mice abdominal cavity, take off the neck executed after 3 d animal, aseptic manipulation in mice abdominal cavity fluid, centrifugal collect macrophages, mixed with LSC - 1 cell culture, after 3 d in liquid, abandon to macrophages.
After a week's interval, the macrophages should be cultured once again.
3) method of inoculation in nude mice:
The LSC - 1 cell press 1 * 106 / mL axillary subcutaneous inoculation of nude mice, continuous observation, when the mass to about 1 cubic centimetres long, take off the neck to be put to death animals, aseptic operation take out the mass, organization of law make the cell and tissue culture.
Effect appraisal
Immunohistochemical identification:
The lsc-1 cells treated with the above three methods were grown in a sterile circular cover with a diameter of 15 mm, and then the keratin AE1 / AE3 immunohistochemical staining and HE staining were made from the wall
Cell cycle identification:
The fluorescence intensity of the cell was measured by flow cytometry, and the DNA content was analyzed.
The lsc-1 cell cycle was tested in three methods and compared with the third-generation cell condition in the early stage of the cell.
Antibiotic withdrawal observation:
The cells of three methods were cultured with the complete culture culture without antibiotics, and 3 weeks of continuous observation were taken to remove the bacteria culture from the cells.
Maintenance, freezing and recovery of cell lines:
The cells that had been cultured without antibiotics for 3 weeks were frozen in a conventional manner and recovered after 1 month to observe the cell growth status.
Results
Self-built people transmit the LSC laryngeal cancer cell line 1 to 12 generations later, cell growth slowing down and wall were post go round, levitating, cell death, cell gap arises to movement of the tiny creatures, but still clear and not cloudy broth, charging that centrifugal supernatant fluid, the sediment routine blood AGAR culture, 24 h the results for sterile growth, began training after 5 ~ 7 d gray small colony, smooth and moist, with transparent ring of hemolysis, automatic bacteria identification device article biochemical identification detection for eosinophilic malt oligotrophic food, bacterium of the bacteria is a gram-negative bacterium fermentation.
Bacterial drug susceptibility results: in 15 antibiotics, the bacteria were only sensitive to ceftidine, pericillin, cefoperazone/sulbatam, and the rest were resistant to drugs.
2.1 antimicrobial effect of antibiotics
After screening, the optimal antimicrobial concentrations of the antibiotics for lsc-1 cells were: pipericillin (64 mg/L), cephalosporin 2 mg/L, cefoperazone/sulbatan 32 mg/L.
The lsc-1 cells were passed from one generation to the other in pipericillin and cefazidine in the best antimicrobial concentration, 3 times in cefoperazone/sulbatam, and 3 in each cell.
The recovery situation: the three strains of pipericillin were not adhered to the wall, and the cephalosporine had been recovered and adhered to the wall, but after a week of growth, the bacteria were found and the cells died.
Another cold storage, after the recovery of a good growth, but bacterial after the withdrawal of antibiotics, was identified as the malt oligotrophic bacteria.
The cefoperazone/sulbatan 3 strains survived, but were slow to grow, and once a week, there was still a small amount of bacteria, and bacterial contamination after the withdrawal of antibiotics.
Among the three antibiotics, cefoperazone/sulbutamine had the best antibacterial action and cell growth status, and cefazidine had a greater damage to the cell. The cells had a rough surface and poor condition.
2.2 antibiotic combined macrophage phagocytosis effect
Compare cephalosporin ketone of pp/shu ba and macrophage successively used and alternate use of two antibacterial effect, and observe the number of batches after recovery, cells cryopreserved specific means is: cefoperazone/shu ba jotham 32 mg/L after training 1, 2, 4 times, then respectively with macrophages trained twice, the cryopreserved 1 strains;
The cefoperazone/sulbatan and macrophages were alternately cultured for 3 times and one strain was frozen.
Cell status after resuscitation: cefoperazone/sulbatan and macrophages were successively used, and the number of cells with more antibiotics was more frequent (5 generations).
The two cells were better than the first 3 cells and had the highest number of generations (6 generations).
However, all cells die due to poor state or reproduction.
The effect of inoculation method in nude mice
After 40 d of the tumor cells were inoculated into the nude mice, the tumor began to emerge, and 60d to about 1 cm3.
The animals were removed from the mass, and the mass of the tumor was 0.39 g, with a volume of 0.8cm * 0.6cm * 0.4cm.
The tissue block was cultured in a complete medium containing tri-reactance. After 14 d, the tissue was grown without antibiotics, and the cells grew well in 3 weeks.
After the natural transfer 8 times, frozen for 1 month after the recovery, the cells can still stick the wall growth.
The bacteria culture was taken and the result was sterile growth.
2.4 cell morphology and biological characteristics
The lsc-1 cell morphology of the antibiotic treated with nude mice was basically the same as that of lsc-1, the cells were polygonal and had the feeling of paving stone.
Immunohistochemical cytokeratin staining, nude mice vaccinated and antibiotic treatment of the cells were AE1 / AE3 positive expression, but positive proportion to the third generation of cells close to the former, the latter less positive expression.
HE staining, the lsc-1 cells inoculated with nude mice were highly heterologous: cell morphology and size;
Nuclear is pleomorphic: size, shape and different dyeing, nucleus and cytoplasm ratio increases (close to 1 b1), nucleolus bulky, populations, fission like increased, appears asymmetric, multipolarity pathological fission, occasional megakaryocyte, cytoplasmic glycogen particles, is consistent with the third generation of cells form LSC - 1.
2.5 cell and cell proliferation cycle analysis results
After treating 3 times with cefoperazone/sulbatan, the lsc-1 cell cycle histogram is a nearly diploid image, but the S stage and G2M phase cell peak are high and still have the characteristics of cancer cells.
The lsc-1 cells were not euploid, and the diploid and heteroploid S phase and G2M period were higher, and were closer to the distribution of the DNA content of lsc-1 cell cycle.
Three kinds of cells each karyotype proportion: nude mice inoculated cells remain diploid and heteroploid karyotype, respectively 44. 92% and 55. 08%, and the third-generation cell (diploid 41. 52%, heteroploid 58. 48%) are basically the same, and cefoperazone/shu and processing of all cells of diploid, different times cells disappeared.
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