Aging Aging

Human T-Cell Clones 55quality IL-2; the investigator should pretest a batch for suitability for thecells being cultured. Mixtures of TCGFs may be useful in some circumstances,especially IL-2 + IL-4 or IL-2 + IL-7 (see Subheading 3.). Major suppliersare Genzyme, Endogen, PeproTech, R and D Systems, Boehringer-Mannheim,and so on.2. Monoclonal antibodies (MAbs): Again, there are many suppliers of different antibodies,and companies favored will be different in different parts of the world.Major suppliers are Becton-Dickinson, Coulter-Immunotech, DAKO, and so on.However, for certain purposes, such as cell isolation with magnetic beads (seeSubheading 2.3.1.), it may be economically desirable to obtain hybridoma cellsand produce MAb oneself. The hybridoma cells are easy to grow, and for thepurposes of cell separation, culture supernatants do contain enough MAbs.Obtaining hybridomas may be a problem, but cell banks such as the AmericanType Culture Collection (ATCC) can provide hybridomas secreting MAbs againstcommon antigens sufficient for most cell separation purposes.3. Magnetic beads:a. Dynabeads (Dynal, Oslo, Norway): In this method, the T-cell population isnegatively selected after the cells are labeled with cocktails of MAbs againstB cells (e.g., CD19), natural killer (NK) cells (CD16), monocytes (CD14),major histocompatibility class (MHC) II, etc. Because of the amount of MAbsrequired it is recommended that hybridoma supernatants are used. DynabeadsM450 coated with sheep anti-mouse IgG can be employed for most negativecell separations.b. Miltenyi CD34 Progenitor Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany)is supplied with the necessary reagents for CD34 cell isolation by positiveselection. The magnetic particles are precoated with CD34 MAbs directedagainst a particular exposed epitope of CD34 (class II epitope); purity of thederived population can then be checked with a MAb directed at a differentepitope (e.g., anti-class I MAb My10 from Coulter-Immunotech).4. Culture medium: Human serum for supplementing media such as RPMI 1640or IDMEM to support long-term growth of T-cells cannot be reliably obtainedcommercially. It is necessary to prepare and screen the serum on T-cells in thelaboratory. Serum can be obtained or purchased from blood banks, but it isdifficult to obtain enough male nontransfused AB donors for regular use. Itmay be satisfactory to use male nontransfused donors of any blood type, as wedo. Serum is separated from coagulated blood by centrifugation and one aliquotfrom each of at least 20 sera prepared at the same time is heat inactivated(30 min at 56°C). The bulk of the sera are frozen separately. The test samplesare then separately tested for their ability to support T-cell proliferation. Thosethat are judged satisfactory are then thawed and pooled. Culture medium mustbe supplemented with 10–20% of such serum pools to support long-term T-cellgrowth.Alternatively, use X-Vivo 10 or X-Vivo 15 serum-free medium (BioWhittacker),formula unknown.