Cell culture and cell isolation protocols  Cryopreservation of CD4+ CellsA part of the cells cultivated in the above item (8) were cryopreserved once on the sixth day of the cultivation. In a method for preservation, the cells adhere to the bottom of the flask were detached by tapping the flask several times, then 500 microl of the culture medium in the flask was taken out and spread onto a tryptic soy agar (TSA) medium, and then incubated in the CO 2 incubator at 37 degree C. to carry out an sterility testing. Furthermore, 10 microl of the culture medium in the flask was taken out and the cell number was counted similarly to the above item (3).Next, 40 ml of the cultured cell solution was transferred to each of two 50 ml tubes, and they were centrifuged at 1,000 rpm at 20 degree C. for 5 minutes. In every tube, the supernatant was discarded and the cell pellet was vortexed, the resulting cell suspension and cell preservation solution [prepared by mixing 5 ml of human serum, 5 ml of dimethyl sulfoxide (hereinafter, abbreviated to DMSO) and a 40 ml of medium (RPMI1640+7)] were cooled down on ice for 5 minutes, the cell suspension of every tube was brought together to one tube with 4.5 ml of the cell preservation solution, the cells in the tube were homogenized by pipetting lightly, and then the resulting matter was divided into three cryopreservation tubes (2.0 ml), respectively. Furthermore, the tubes were put in a BICELL (Nihon Freezer co. Ltd., Tokyo Japan), a freezing container, and preserved at -80 degree C. for several days, and then preserved in a liquid nitrogen tank. The cell concentration at the culture was 1.9times10 6/ml, and the preserved cell number was 5.1times10 7/tube.
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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