As shown in
The preferred embodiments according to the present invention are illustrated hereinafter.
Ultra-high molecular weight polyethylene particles having particle size of 3.3 μm were added to a plate and were suspended in the culture medium to form a suspension of ultra-high molecular weight polyethylene particles. A small amount of the cell suspension was taken and dripped into a blood cell counting plate. The cells in the cell suspension were counted under an optical microscope, thereby determining the density of the ultra-high molecular weight polyethylene particles in the cell suspension.
J774A.1, a murine macrophage line, was cultured in DMEM medium containing 10% fetal bovine serum. After grown to confluence, the cells were washed with PBS one or two times, scraped off the plate with a blade and then suspended in PBS. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was removed. The cells were re-suspended in fresh medium. A small amount of the cell suspension was taken and dripped into a blood cell counting plate. The cells in the cell suspension were counted under an optical microscope, thereby determining the density of the cells in the cell suspension. One milliliter of medium and 3×105 cells were added to a 24-well plate and incubated in a incubator containing 5% CO2 at 37° C. for 24 hours. After the cells adhered to the interior bottom surface of the plate, the old medium was replaced with 3 ml of fresh medium and a suspension of ultra-high molecular weight polyethylene particles was then added to fill up the well. The number ratios of the ultra-high molecular weight polyethylene particles to the cells were adjusted to 0:1, 0.1:1 and 1:1, respectively. The plate was covered with a treated polyvinyl chloride film, and then carefully inverted. The inverted plate was incubated in an incubator containing 5% CO2 at 37° C. for 5 days.
After culture was finished, the medium in the plate was drained out, then 100 μl of 0.2% MTT solution was added to each well of the plate under light-shielded condition and the plate was incubated at 37° C. for 3 hours. MTT solution was drained out and 200 μl of DMSO was added to each well. After the plate was shaken for 15 minutes, 100 μl of the sample was taken out from each well and added to an ELISA plate. The absorbance at 570 nm of the sample was determined by using an ELISA reader and the cell viability was calculated from the absorbance values. The results were shown in
In this Example, the concentration of cytokine TNF-α was measured by an ELISA method. A capture antibody was added to the wells of an ELISA plate and incubated overnight. Next day, the capture antibody was drained out. After washing three times, a blocking buffer was added and incubated for 1 hour. After washing 3 times, the medium that had been used for culturing the cells was added and incubated for 2 hours. After washing 3 times, a detection antibody was added and incubated for 2 hours. After washing 3 times, streptavidin-HRP was added and incubated for 20 minutes in a dark place. After washing 3 times, a substrate solution was added and incubated for 20 minutes in a dark place. A stop solution was then added. The absorbance at 450 nm of the sample was measured using an ELISA reader and the concentration of TNF-α was calculated from the measured absorbance value. The results under different ratios of particle number to cell number were shown in
As stated above, the present invention complies with the three requirements for a patentable invention: novelty, inventive-step (non-obviousness) and industrial utilization. The present invention has been disclosed by above preferred Examples. However, the persons skilled in the art should understand that these Examples just illustrate the present invention but by no means limit the scope of the present invention in any way. It should be also noted that any modification and replacement having equivalent effects to these Examples are considered falling into the scope of the present invention, and the scope of the present invention is indicated by the following claims.