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1. Harvest cells from a culture that is at or near peak density by centrifugation at 600 x g for 5 min. Pool the cell pellets into a single tube.
2. Adjust the concentration of cells to 2.0 x 10⁶/mL.  If the concentration is too low, centrifuge at 600 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
3. Prepare a 15% (v/v) sterile DMSO solution in ATCC medium 1034 as follows:
   1. Add the required volume of DMSO to a glass screw-capped test tube and place on ice.  Allow the DMSO to solidify.
   2. Add the required volume of refrigerated ATCC medium 1034.  Dissolve the DMSO by inverting several times.  If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 10⁶ and 7.5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process should be no less than 15 min and no longer than 60 min.
5. Dispense in 0.5 mL aliquots into 1.0 mL to 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen.
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just sufficiently to cover only the frozen material. Do not agitate the vial.
9. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate into a fresh tube or flask of ATCC medium 1034.
10. Incubate at 25°C with the cap screwed on tightly (incubate a test tube on a 15° horizontal slant).

Clark CG, Cross GA. Circular ribosomal RNA genes are a general feature of schizopyrenid amoebae. J. Protozool. 35: 326-329, 1988. PubMed: 2840492

Clark CG, Cross GA. rRNA genes of Naegleria gruberi are carried exclusively on a 14- kilobase-pair plasmid. Mol. Cell. Biol. 7: 3027-3031, 1987. PubMed: 2823115

De Jonckheere JF. A century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 41: 309-342, 2002.

Fulton C. Cell differentiation in Naegleria gruberi. Annu. Rev. Microbiol. 31: 597-629, 1977. PubMed: 334046

Fulton C, et al. Chemically defined media for cultivation of Naegleria gruberi. Proc. Natl. Acad. Sci. USA 81: 2406-2410, 1984.

Fulton C. Methods in cell physiology. vol. 4New York: Academic Press; 1970.

Simione FP Jr., Daggett PM. Freeze preservation of pathogenic and nonpathogenic Naegleria species. J. Parasitol. 62: 49, 1976. PubMed: 1255382

Simpson AGB, et al. The evolutionary history of kinetoplastids and their kinetoplasts. Mol. Biol. Evol. 19: 2071-2083, 2002. PubMed: 12446799

Nucleotide (GenBank) : M18732 N.gruberi 18S subunit ribosomal RNA gene.