Cell culture and cell isolation protocols  Harvesting of CD4+ Cells1000 ml of the culture medium cultivated in the bag in the above item (12) was transferred in 250 ml centrifugation tubes aseptically, and the tubes were centrifuged at 1,500 rpm at 20 degree C. for 8 minutes. After the centrifugation, the supernatant was discarded, the cell pellet was loosened with vortex mixer, 250 ml of the cell washing solution was put into the centrifugation tube, and then it was centrifuged at 1,800 rpm at 20 degree C. for 8 minutes. After the centrifugation, the supernatant of it was discarded and the cell precipitate was loosened by vortexing. 100 ml of the cell washing solution was put into the sedimentation tube, each of the cell suspension was transferred into two 250 ml tubes, and then they were centrifuged at 1,800 rpm at 20 degree C. for 8 minutes. After the centrifugation, the supernatant was discarded, the cell precipitate was loosened by vortexing, and then 50 ml of the cell washing solution was put into the two 250 ml tubes respectively.After the centrifugation, the supernatant was discarded and the cell pellet was loosened well with vortex mixer. The cells were suspended in a final solution (a solution for administering lymphocyte was made by putting 100 ml of saline and 5 ml of 20% human serum albumin solution into a 250 ml centrifugation tube to be a final solution) sufficiently, it was passed through a filter with 100 micro-mesh and then fluid volume of the cell suspension was measured.Besides, the cell suspension was sampled in order to count the cell number and the content of CD4. 50 ml syringe was connected with a separation bag (300 ml) to pour the cell suspension through the syringe into the separation bag. When the pouring was completed, liquid remained in the syringe was pushed out by an inner cylinder, and finally the tube was clamped by a forceps. The tube was sealed and syringe was cut off by scissors. As a result of the measurement, the liquid volume was 100 ml and the total cell number was 3.7times10 9.
Source: 20110097314
Disclaimer: Sciclips does not guarantee the authenticity or validity of the protocols listed in this section. These protocols are extracted "as is" from various sources, with minor modifications. Please refer to the respective citations, which are hyperlinked, for more details. The use of cited molecules, not limited to proteins, nucleic acids, compounds etc, in the protocols that are extracted from patents may be covered under patent rights. These molecules should be used for information purpose only.
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