Ahquot 0 34.5 mL into cryovlals Store at -20☌ + S’C. PHA stock solution: Reconstitute PHA (10 mg bottle) with 10 mL RPM1 1640.
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Isolation and Expansion of HIV from CocultureĢ Complete medium: RPM1 1640, 15% FBS, 10% IL-2,1% Pen-strep, 2 rnA4 L-Glutamine Filter sterilize using 0 2-punit. Filter stertltze using a 0.2-u filtration unit. Ftcoll wash medium: RPM1 1640,2% FBS, 1% Pen-strep. PHA (Murex cat #HA 17 or equtvalent, 10 mg lyophyhzed powder) L-Glutamme (200 mM) (Grbco or equivalent) IL-2 (Boehrmger-Mannheim cat# 663-58 1, or equivalent). RPM1 1640 (Gibco or equivalent) Fetal bovine serum (FBS) (Gibco or equivalent) (heat Inactivated, 56☌ 30 mm) Pen-Strep (Gibco or equivalent) Concentratton = 10,000 U Pen and 10 mg strep/ml Ficoll-Paque (Pharmacia or equivalent). In this work, H9 cells are used as an example. There are numerous cell lines available through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH) suitable for HIVinfection and expansion of virus. To obtain the PBMCs from the leukopacks, follow the same procedure described for patient whole blood samples (Subheading 3.1.) 2.2. The donors should be screened by the standard assayscurrently required by the Food and Drug Administration (FDA) and the American Association of Blood Banks (AABB). Donor PBMC Normal donor buffy coats (leukocyte-enriched whole blood, from an HIV negative donor), also known as leukopacks, may be obtained from a blood bank or donor service for processing within 12 h of collection. Although vu-us isolation and expansion can also be performed using contmuous cell lmes, the focus of this chapter will be with the use of PBMCs.
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The establishment and culturing procedure for isolating and expanding HIV-l from a variety of patient specimen types is described below. This practice of standardizing assaycoculture conditions not only muumizes experimental variation but also limits the opportutnty for genetic drift of viruses during prolonged expansion periods and the use of PBMCs from multiple donors. In the author’s laboratory, PBMCs from these selected donors are routinely cryopreserved and reserved specifically for studies. This pro-įrom Methods m Molecular Medrcme, Vol by N L Mtchael and J H Kim 0 HumanaĬess is facilitated by utilizing target PBMCs from donors prescreened for effcient virus isolation and expansion. To mmimize genetic drift (selection) during the periods of virus isolation, it is crttical that these procedures be completed as quickly as possible. Furthermore, these authors also noted that the time required for virus isolation decreased with disease progression. (1) demonstrated vn-us isolation rate increases with CD4 cell depletion and impaired delayed hypersensitivity. Isolation rates reported for patients with AIDS are consistently higher than from pre-AIDS patients Using the Walter Reed classification system, Burke et al. In addition, disease state also influences the isolation of vnus. Patient PBMCs represent a useful source of patient vnus, however, isolates can be obtained from such body fluids as spinal fluid or milk.
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It is well recognized that the efficiency of virus isolation is dependent on specimen type. Cultures fatling to demonstrate evidence of vnus expression within 35 d are usually terminated. Durmg this cocultivation period, culture fluids are harvested at regular intervals and tested for the presence and subsequent replication of HIV. These cocultures are then maintained by regularly scheduled mterleukin-2 (IL-2) supplemented medium replacement, and the periodic addition of freshly stimulated normal donor PBMCs. This may be achieved by the coculttvation of pattent material with mitogen-stimulated peripheral blood mononuclear cells (PBMCs) from normal, healthy donors. Typically, the quantity of biologically active virus, or viral protein, in body tissues is below the level of direct detection by either antigen capture or reverse transcrtptase assays.Consequently, the virus must be expanded in culture. Introduction HIV can be recovered from infected patients at all stagesof the disease spectrum. 1 Isolation and Expansion of HIV from Cells and Body Fluids by Coculture James R.