Cell Biology Techniques

I.  Detection techniques

      A.     Radiolabeling

          1.  Use of various radioactive isotopes inserted into chemicals used by cells

                2.  Autoradiography (use of film to detect radio-decay from isotopes)

                3.  Scintillation counter-used to count radio-active decay (quantify radioactivity)

      B.  Immunocytochemistry

   1.      Use of specific primary antibodies to detect specific antigen (compound that you want to detect)

                2.  Use secondary antibodies (bound to chromogen) to amplify primary signal

II.  Cell cultures

      A.  Primary cell culture

               1.  extracted from tissue

               2.  finite life span

      B.  Immortalized cells

               1.  obtain from vender or  formed “spontaneously”

               2.  infinite life span (many derived from cancerous tissue)

      C. Limits to cell culture

               1.  media

               2.  contamination (must use sterile technique)

               3.  confluence (contact inhibition)

               4.  application to in situ tissue?

III.  Separation techniques

      A.  Centrifugation

                1.  separates chemicals/organelles by size/density

                2.  sedimentation coefficients used to identify

      B.  Chromatography

                1.  use of platform (paper, TLC, column, gel, etc.) to separate chemicals

                2.  resolution by size, charge, binding properties

                3.  HPLC

IV.  DNA Technology

      A.  Purify DNA

      B.  Cut/Isolate DNA fragments

                 1. Restriction endonucleases (cut known palindromic DNA sequences)

                 2.  Reverse transcriptase (mRNA into DNA)

     3.  PCR-polymerase chain reaction-use of heat-stable Taq polymerase to make multiple    

          copies of known DNA pieces

      C.  Monitor DNA recombination

                 1.  Electrophoresis-Southern, Northern, Western blots

     2.      DNA sequencing (Sanger method)-use of dideoxynucleotides to identify DNA sequence

                 3.  Microarrays-detection of gene expression

      D.  DNA transfection

                1.  Bacteria plasmids (use restriction enzymes to cut both plasmid and foreign DNA)

                2.  Plant transfection-Agrobacterium tumefaciens

                3.  Mammalian cells-viruses (SV-40, adenovirus)