Aging Aging

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300 Grubeck-Loebenstein, Saurwein-Teissl, and Romani10 min. Wash twice in culture medium afterwards. At this point the cells mayeither be processed further to enrich LCs (step 5a) or be put in culture (step 5b).5a. To preenrich LCs, epidermal cells can be treated with anti-Thy-1 and complementto remove most keratinocytes and lymphocytes. In a 50-mL polypropylenetube, resuspend 100–150 × 10 6 epidermal cells (the expected yield from 60 ears)in 3 mL of hybridoma culture supernatant of MAb anti-Thy–1 (ATCC no. TIB99).Then add 10 mL of a sterile-filtered solution consisting of 8.5 mL of cytotoxicitymedium, 1 mL of reconstituted complement, and 0.5 mL of DNase stock solution.After incubation for 1 h at 37°C in a shaking water bath, the resulting epidermalcell suspension (viability 10–20%) is washed twice with cold PBS. Treatcells for 10 min at 37°C with 0.125% trypsin and 80 µg/mL of DNase in PBS at acell concentration of 1–2 × 10 6 viable cells/mL. To stop the digestion, add anequal volume of culture medium and centrifuge the cells for 10 min at 4°C, at200g with brakes off. This simple procedure (“trypsin trick”) removes most deadcells and results in viable (>90%) epidermal cell suspensions containing about15% (range 10–28%) LCs, which can then be further purified by positive selectionby sorting (by FACS or MACS, Miltenyi, 1990) for, for example, MHC classII (see Note 3).5b. Untreated or anti-Thy–1/C' treated (i.e. LC preenriched) epidermal cell suspensionscan be cultured for 2–4 d after which the LCs have matured and can beenriched to 60–90% by simple Ficoll–Hypaque/Lymphoprep density gradientcentrifugation according to standard methodology of the nonadherent fractionand harvesting of the low-density fraction (see Note 4).3.2. Purification of DCs (LCs) from Human Skin1. Incubate skin for about 1 h in culture medium containing 25 mM HEPES and10-fold concentrated gentamicin at room temperature (see Note 5).2. Rinse the pieces of skin twice in PBS in 100 mm Petri dishes. Cut it into pieces(1 cm 2 if thin, 15 mm 2 if thick) and put them into another 100 mm Petri dishcontaining 11.25 mL of PBS. When correctly placed epidermal side up, the skinwill spread on the PBS. When all pieces are floating on PBS add 1.25 mL of 2.5%trypsin.3. Incubate with trypsin overnight at 4°C.4. Next morning aspirate the medium from the dishes and remove epidermal sheetsfrom the dermis with two thin forceps. Put epidermal sheets into a tea strainerplaced in another 100 mm Petri dish with 30 mL of culture medium containing80 µg/mL of DNase I and 25 mM HEPES buffer. The sheets should not overlap.When the strainer is filled with sheets, stir vigorously to release single cells,remove the strainer from the dish, filter the cell suspension through a Nitex nylonmesh (54 µm mesh size), and centrifuge it in 50-mL tubes at 300g for 10 minfollowed by two washes in culture medium. The primary epidermal cell suspensioncontains between 1% and 3% LCs (see Note 6).5. Culture of LCs: Plate epidermal cells in culture medium in culture vessels ofyour choice.
Dendritic Cells in Old Age 3016. Harvest of cultured LCs: After 3 d of culture nonadherent cells are rinsed off thetissue culture vessels and collected. This cell suspension has a very low viability(sometimes
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6 Strehlerlead to possible death si
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Understanding Aging 9the intestine
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Understanding Aging 117. Do long-li
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Understanding Aging 13is, the great
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Understanding Aging 15While this re
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Understanding Aging 172. Pearl, R.
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Understanding Aging 1945. Perovic,
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24 Cristofalo, Volker, and Allenmen
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26 Cristofalo, Volker, and Allenrec
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28 Cristofalo, Volker, and Allenski
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30 Cristofalo, Volker, and AllenTab
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34 Cristofalo, Volker, and AllenNa
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38 Cristofalo, Volker, and Allentak
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40 Cristofalo, Volker, and Allenb.
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42 Cristofalo, Volker, and Allenand
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44 Cristofalo, Volker, and AllenRef
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46 Cristofalo, Volker, and Allen33.
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48 Cristofalo, Volker, and Allen61.
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50 Cristofalo, Volker, and Allenhum
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52 Cristofalo, Volker, and Allen125
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54 Pawelecdiluted cells on an irrad
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56 Pawelec3. Method3.1. Source of C
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58 Pawelecweekly or fortnightly sub
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60 Pawelection, the VERUM Foundatio
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Telomeres and Replicative Senescenc
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Detection of Molecular Events 715De
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Detection of Molecular Events 73hav
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Detection of Molecular Events 75Fig
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Detection of Molecular Events 772.
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Detection of Molecular Events 79Fig
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86 Kirk and Millerexperiments on un
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88 Kirk and MillerFig. 1. (A) Analy
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90 Kirk and MillerFurthermore, limi
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92 Kirk and Millersufficiently dilu
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94 Kirk and Miller4. Run samples on
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96 Kirk and Miller16. Lange-Carter,
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98 Engel, Adibzadeh, and PawelecPCR
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100 Engel, Adibzadeh, and Pawelec5.
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102 Engel, Adibzadeh, and PawelecTa
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104 Engel, Adibzadeh, and PawelecTa
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106 Engel, Adibzadeh, and Pawelecam
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110 Engel, Adibzadeh, and Pawelecfo
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114 Engel, Adibzadeh, and Pawelecha
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Xenobiotic-Metabolizing Enzymes 119
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Assessing Antioxidant Status 1339As
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Assessing Antioxidant Status 135Fig
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Assessing Antioxidant Status 137Tab
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Assessing Antioxidant Status 139add
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Assessing Antioxidant Status 14121.
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Comet Assay of DNA Damage 14310Meas
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Comet Assay of DNA Damage 145contro
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Comet Assay of DNA Damage 1476. Lys
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Comet Assay of DNA Damage 1494. Gen
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Comet Assay of DNA Damage 151anode
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Comet Assay of DNA Damage 1535. As
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Comet Assay of DNA Damage 15515. In
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Comet Assay of DNA Damage 15720. Co
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160 van der Schanswithin 1 h, after
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162 van der Schans10. High-binding
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164 van der Schans3. Neutralize aft
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166 van der SchansThe calculations
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8-oxoguanine Levels in Nuclear DNA
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Mutation and the Aging Process 1791
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Mutation and the Aging Process 181m
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Mutation and the Aging Process 183b
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190 HouThe HPRT mutational spectrum
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192 Houamplifications are carried o
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194 Hou4. Reading: Load 10 µL of t
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196 Hou4.2. MP-PCRUse preferably DN
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Susceptibility of LDL to Oxidation
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Analysis of Pentosidine 20916Measur
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Analysis of Pentosidine 211Fig. 2.
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Analysis of Pentosidine 213Fig. 4.
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Analysis of Pentosidine 2152. Preci
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Analysis of Pentosidine 2177. Nagar
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222 Miquellipoperoxides and malonal
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224 Miquelat 4°C. Protect this sol
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226 Miquel3.1. Animal AnesthesiaMed
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228 Miquelsteps to carry out a conv
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230 Miquelsuspending it over the so
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232 Miquelcontrast, washing with bu
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234 MiquelFig. 3. Electron microsco
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Damage to Mitochondria 23718Causes
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Damage to Mitochondria 239Fig. 1. E
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Damage to Mitochondria 2412. To obt
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Damage to Mitochondria 243synthesis
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Mitochondrial DNA Mutations 24519An
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Page 260 and 261: 282 BeckmanWhatever the mechanism e
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Page 288 and 289: NK Cell Function in Aging 31123Age-
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354 Yuto-implement intervention and
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356 Yuof their quality control proc
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358 Yugenesis of major diseases (1-
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Genetically Engineered Mice 36126Th
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Genetically Engineered Mice 363as t
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Genetically Engineered Mice 365in b
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Genetically Engineered Mice 367numb
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Genetically Engineered Mice 369mula
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Aging Aging

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