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Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.  Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.  
5. Resuspend cells in fresh growth medium.  Add appropriate aliquots of cell suspension to new culture vessels.
6. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommended

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Deleersnyder V, et al. Formation of native hepatitis C virus glycoprotein complexes. J. Virol. 71: 697-704, 1997. PubMed: 8985401

Rhoades KL, et al. Synergistic up-regulation of the myeloid-specific promoter for the macrophage colony-stimulating factor receptor by AML1 and the t(8;21) fusion protein may contribute to leukenogenesis. Proc. Natl. Acad. Sci. USA 93: 11895-11900, 1996. PubMed: 8876234

Halford WP, et al. Mechanisms of herpes simplex virus type 1 reactivation. J. Virol. 70: 5051-5060, 1996. PubMed: 8764012

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Berson JF, et al. A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus tyep 1 strains. J. Virol. 70: 6288-6295, 1996. PubMed: 8709256

Goodrum FD, et al. Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells. J. Virol. 70: 6323-6335, 1996. PubMed: 8709260

Kolanus W, et al. alphaLbeta2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1 a cytoplasmic regulatory molecule. Cell 86: 233-242, 1996. PubMed: 8706128

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Standard Test Method for Determining the Virus-Eliminating Effectiveness of Liquid Hygienic Handwash and Handrub Agents Using the Fingerpads of Adult Volunteers. West Conshohocken, PA:ASTM International;ASTM Standard Test Method E 1838-02.

Standard Test Method for Evaluation of Handwashing Formulation for Virus-Eliminating Activity using the Entire Hand. West Conshohocken, PA:ASTM International;ASTM Standard Test Method E 2011-99.

Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides. West Conshohocken, PA:ASTM International;ASTM Standard Test Method E 2197-02.

Nucleotide (GenBank) : U83324 Cercopithecus aethiops CC chemokine receptor-5 (CCR5) gene, clone 1, complete cds.

Nucleotide (GenBank) : U83325 Cercopithecus aethiops CC chemokine receptor-5 (CCR5) gene, clone 2, complete cds.