Aging Aging

NK Cell Function in Aging 3135. Petri dishes (Corning Costar Europe, Bad Loevedorp, The Netherlands).6. Nylon wool (Robbins, Sunnyvale, CA, USA).7. Monoclonal antibodies (MAbs): anti-CD16 (3G8, Immunotech, Marseille Cedex,France; Leu11, Becton Dickinson, San Josè, CA, USA), anti-CD56 (NKH-1,Coulter Corporation, Miami, FL, USA; Leu19 Becton Dickinson, San Josè, CA,USA), anti-CD94 (HP3B1, Coulter Corporation, Miami, FL, USA). These MAbsare available as PE or FITC labeled. Double fluorescence with anti-CD3 (Anti-Leu 4, Becton Dickinson, San Josè, CA, USA) is recommended.8. For intracellular labeling of perforins the following additional material isrequired:a. 2% Paraformaldehyde (Fluka Chemie AG, Buchs, Switzerland) solution inPBS.b. 0.2% Tween-20 (Sigma, St. Louis, MO, USA) solution in PBS.c. PBS with 2% FCS and 0.1% sodium azide (Sigma, St. Louis, MO, USA)(PBS–FCS–Az).d. MAb anti-human perforin and Ig isotype control (Pharmingen, San Diego,CA, USA).e. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugatedgoat anti-mouse Ig (GAM-FITC or GAM-PE) (Pharmingen, San Diego,CA, USA).f. Normal mouse serum (Dako, Glostrup, Denmark).9. Flow cytometer (FacsCount, Becton Dickinson, San Josè, CA, USA).2.2. NK Cell Enrichment and Purification1. LSM, density 1.077 g/mL (Histopaque–1077, Sigma, St. Louis, MO, USA).2. Culture medium prepared as indicated in Subheading 2.1.3. PBS (Gibco Life, Paisley, UK).4. Laminar flow hoods.5. Petri dishes (Corning Costar Europe, Bad Loevedorp, The Netherlands).6. Nylon wool (Robbins, Sunnyvale, CA, USA).7. MAbs: anti-CD3 (Anti-Leu 4, Becton-Dickinson, San Josè, CA, USA) anti-CD19(J4.119, Immunotech, Marseille Cedex, France; Leu12 Becton Dickinson, SanJosè, CA, USA), and anti-CD16 (3G8, Immunotech, Marseille Cedex, France;Leu11, Becton Dickinson, San Josè, CA, USA).8. GAM coupled magnetic beads (Dynal AS, Skoyen, Norway).9. Magnetic particle concentrator (MPC1, Dynal AS, Skoyen, Norway).10. MACS cell separation reagents and equipment (Miltenyi Biotec GmbH, BergischGladbach, Germany) can also be used to purify NK cells.a. Separation reagents: the NK isolation kit consists of: reagent A, a cocktail ofhapten-conjugated monoclonal CD3, CD4, CD19, CD33 antibodies; reagentB, colloidal superparamagnetic MACS microbeads conjugated to an anti-haptenantibody and depletion columns.b. Equipment: VarioMACS magnetic cell separator.