1. Mix the components in the order listed. When the medium is added to the DMSO the solution will warm up due to chemical heat.
2. Harvest cells from a culture that is at or near peak density by filtration and centrifugation at 800 x g for 5 min.
3. Adjust the concentration of cells at least 2 x 10⁶/mL in fresh medium.
4. Mix the cell preparation and the cryoprotective solution in equal portions.
5. Dispense in 0.5 mL aliquots into 1.0 mL toxa0 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. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.) xa0
7. Ampules are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place the vial in a 35°C water bath.xa0 Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial. Immediately after thawing, do not leave in water bath, aseptically remove the contents of the ampule and inoculate into a T-25 tissue culture flask containing 10 mL ATCC medium 1525 bacterized with Klebsiella pneumoniae subsp. pneumoniae (ATCC^® 700831) or Enterobacter aerogenes (ATCC^® 13048).
9. Incubate at 25°C with the cap screwed on tightly.
10. Once the culture is established, vigorously agitate the flask and aseptically transfer 0.5 mL to 10.0 mL of bacterized ATCC medium 1525.
11. Follow the protocol for maintenance of culture.

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Collins AG. Evaluating multiple alternative hypotheses for the origin of Bilateria: An analysis of 18S rRNA molecular evidence. Proc. Natl. Acad. Sci. USA 95: 15458-15463, 1998. PubMed: 9860990

Lang BF, et al. The closest unicellular relatives of animals. Curr. Biol. 12: 1773-1778, 2002. PubMed: 12401173

King N, Carroll SB. A receptor tyrosine kinase from choanoflagellates: molecular insights into early animal evolution. Proc. Natl. Acad. Sci. USA 98: 15032-15037, 2001. PubMed: 11752452

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Nucleotide (GenBank) : AAK27412 actin Monosiga brevicollis.

Nucleotide (GenBank) : AAK27411 beta-tubulin Monosiga brevicollis.

Nucleotide (GenBank) : AAK27410 alpha-tubulin Monosiga brevicollis.

Nucleotide (GenBank) : AAK27414 elongation factor 2 Monosiga brevicollis.

Nucleotide (GenBank) : AY026072 Monosiga brevicollis actin mRNA, complete cds.

Nucleotide (GenBank) : AY026071 Monosiga brevicollis beta-tubulin mRNA, complete cds.

Nucleotide (GenBank) : AY026070 Monosiga brevicollis alpha-tubulin mRNA, complete cds.

Nucleotide (GenBank) : AAL33602 receptor tyrosine kinase MBRTK1 Monosiga brevicollis.

Nucleotide (GenBank) : AAG10297 small subunit ribosomal protein 3 Monosiga brevicollis.

Nucleotide (GenBank) : AAK27413 elongation factor 1 alpha long form Monosiga brevicollis.

Nucleotide (GenBank) : AAK27415 elongation factor 1 alpha short form Monosiga brevicollis.

Nucleotide (GenBank) : AY026074 Monosiga brevicollis elongation factor 2 mRNA, complete cds.

Nucleotide (GenBank) : AF440359 Monosiga brevicollis receptor tyrosine kinase MBRTK1 mRNA, partial cds.

Nucleotide (GenBank) : AAK00311 DNA-dependent RNA polymerase II largest subunit RPB1 Monosiga brevicollis.

Nucleotide (GenBank) : AF440360 Monosiga brevicollis receptor tyrosine kinase MBRTK1-like protein gene, partial sequence.

Nucleotide (GenBank) : AF315821 Monosiga brevicollis DNA-dependent RNA polymerase II largest subunit RPB1 (RPB1) gene, partial cds.

Nucleotide (GenBank) : AY026073 Monosiga brevicollis elongation factor 1 alpha long form mRNA, complete cds, alternatively spliced.

Nucleotide (GenBank) : AY026075 Monosiga brevicollis elongation factor 1 alpha short form mRNA, partial cds, alternatively spliced.

Nucleotide (GenBank) : AF275274 Monosiga brevicollis small subunit ribosomal protein 3 (rps3) gene, partial cds; mitochondrial gene for mitochondrial product.