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Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
CRYOPRESERVATION:?
??????? Only young cells (rings) can be frozen in glycerolyte medium** because their membranes are more robust.
1. Centrifuge ring stage culture for 5 min at 1800rpm in 50 ml centrifuge tube.
2.???? Aspirate supernatant using sterile Pasteur pipet.
3.???? Resuspend pellet gently in remaining supernatant.
4.???? Slowly add 5 volumes of glycerolyte medium (see below) to 3 volumes pellet dropwise via a syringe as follows:
??????? A.??? Add the first volume of glycerolyte and allow the tube to stand for 5 mins. at room temperature.
B.??? Add the remaining 4 volumes of glycerolyte and gently agitate.
5.???? Aliquot mixture into Nunc screwtop freezing vials and store at ?80oC overnight.
6.???? Plunge vials into liquid nitrogen (-196oC) the next day and store in liquid nitrogen or liquid nitrogen vapor.
** To formulate glycerolyte medium, combine the following with distilled water to 100 ml:? 57.00g glycerol, 1.60g sodium lactate (C3H5NaO3), 30.00mg potassium chloride (KCl), 1.38g sodium dihydrogen phosphate (NaH2PO4). Mix well and adjust pH to 6.8 using concentrated NaOH and/or HCl. Autoclave to sterilize, and store at 4oC.
Trager W, et al. Clones of the malaria parasite Plasmodium falciparum obtained by microscopic selection: their characterization with regard to knobs, chloroquine sensitivity, and formation of gametocytes. Proc. Natl. Acad. Sci. USA 78: 6527-6530, 1981. PubMed: 7031656
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