Detection of apoptosis in cartilage in situ and in isolated chondrocytes.
Apoptosis is a form of programmed cell death conceptually opposed to necrosis. In view of the inherent difficulty in accurately detecting apoptosis in chondrocytes, this chapter describes complementary techniques that may be used in combination. During apoptotic death, protein and DNA breakdown is accomplished by caspases (cysteine proteases) in a highly regulated manner. Activation of caspases occurs in the initiation and/or the execution phase of certain apoptotic programs and represents an early physiologic marker of apoptosis. Here we present an immunoblotting technique that allows the detection of caspase-3 processing in cultured human chondrocytes. Apoptosis leads to plasma membrane asymmetry and to externalization of phosphatidylserine residues, which are bound with high affinity by annexin V. In the early stages of apoptosis, cells typically have an intact cell membrane. Apoptotic cells will not stain positive with propidium iodide, whereas externalization of phosphatidylserine will be detected by annexin V. Terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) works on the principle that DNA strand breaks (single or double) that occur during apoptosis can be identified by labeling free 3'-hydroxyl termini. Labeled nucleotides are polymerized to these termini in a reaction catalyzed by TdT. The tissue can then be examined histologically for identification of TUNEL-positive cells in situ.
D'Lima
Darryl D
DD
Orthopaedic Research Laboratories, Scripps Clinic Center for Orthopaedic Research and Education, La Jolla, CA, USA.
Kuhn
Klaus
K
Lotz
Martin K
MK
D017304:Annexin A5 / Q000737:chemistry; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis; D002356:Cartilage / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D019902:Chondrocytes / Q000737:chemistry* / Q000166:cytology / Q000648:ultrastructure*; D005434:Flow Cytometry; D005944:Glucosamine / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D009599:Nitroprusside / Q000494:pharmacology; D011419:Propidium / Q000737:chemistry; D019311:Staurosporine / Q000494:pharmacology
D017304:Annexin A5; D000906:Antibodies; C481004:CH-11 anti-fas antibody, human; D009599:Nitroprusside; D011419:Propidium; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine; D005944:Glucosamine