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lecture14 - 1 3.051J/20.340J Lecture 14 Quantifying Cell...

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1 3.051 J / 20 .340 J Lecture 14: Quantifying Cell Function c. Cell Proliferation Assays Importance: wound healing immune host defense cancer therapy tissue engineering bioprocessing/synthesis of theraputic glycoproteins ¾ Cell number 1. count via microscopic observation or Coulter counter 2. compute specific cell proliferation rate const : 1 dN d (ln N ) k g = N dt = (units: t -1 ) dt N = # of cells at time t ¾ Cell phase populations In some cases, we want to know the population of cells in each phase of the cell cycle (eukaryotes): M = Mitosis (~ 1 h) G M G 1 = gap between cell division & 2 DNA synthesis (~18-72 h) S = DNA synthesis (~6-8 h) G 1 S G 2 = gap between DNA synthesis & mitosis (~2-3 h) G o = quiescent cells
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2 3.051 J / 20 .340 J tritiated-thymidine uptake 1. cells exposed to [ 3 H]thymidine pulse which labels S-phase cells only 2. % S obtained from autoradiography (Ag precipitates in an overlying emulsion film reveal S-phase cells, similar to a photograph emulsion*) 3. % M and t M obtained from optical microscopy (visually distinct) t M t M t G1 + t S + t G2 t cycle 4. by following % labeled mitoses after [ 3 H]thymidine pulse through 2 cell divisions, t G2 , t S and t cycle can be determined t G1 t cycle t S % labeled t G2 [ 3 pulse *autoradiography is also used with mitoses time H] thymidine PAGE for protein identification
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