1 3.051J/20.340J 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 )kg = Ndt = (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) GM G1 = gap between cell division & 2 DNA synthesis (~18-72 h) S = DNA synthesis (~6-8 h)G1 S G2 = gap between DNA synthesis & mitosis (~2-3 h) Go = quiescent cells2 3.051J/20.340J tritiated-thymidine uptake 1. cells exposed to [3H]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 tM obtained from optical microscopy (visually distinct) tMtM tG1 + tS + tG2 tcycle 4. by following % labeled mitoses after [3H]thymidine pulse through 2 cell divisions, tG2, tS and tcycle can be determined → tG1 tcycle tS % labeled tG2 [3pulse *autoradiography is also used with mitoses time H] thymidine PAGE for protein identification3 3.051J/20.340J flow cytometry* 1. cells’ DNA labeled with DNA-specific fluorescent dye (ex. acriflavine, ethidium bromide) 2. count cell # vs. fluorescence using laser excitation 3. fluorescence intensity ~ DNA present G1:S:G2:M DNA (& fluorescence) ratio is 1:1-2:2:2 G1 S G2+M area % 52 41 7 cell # 0 relative DNA content 100 Advantages: Drawback: - no radiolabeling - M & G2 populations lumped - faster than autoradiography*flow cytometry is also used to determine expression of specific cell receptors by labeling with fluorescent antibodies4 3.051J/20.340J d. Cell Differentiation Assays Importance: - change in phenotype renders specialization of function - characterized by changes in protein synthesis, genes expressed & secretions 2D Gel Electrophoresis (SDS-PAGE): cell proteins separated on basis of mass and charge—providing a “signature” of the cell Method 1. cell is lysed (contents extracted) 2. protein solution is separated by pI (isoelectric point = pH where net charge = 0) using isoelectric focusing pH 9 pH 2 i. ampholyte mixture is placed under E-field to create a stable pH gradient in a polyacrylamide gel. ii. protein solution is added and E-field reapplied iii. proteins diffuse to their isoelectric point 3. gel strip is next placed crosswise on a second gel incorporating sodium dodecyl sulfate (SDS) 4. SDS binds & denatures proteins ⇒ native charge becomes negligible ~1 SDS per 2 amino acids charge/mass ≈ constant5 3.051J/20.340J 5. gel is placed under E-field, separating protein chains via molecular weight low molecular weight species move faster ,µ=µ (cgel , E MWprotein ) SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis MW pI 6. compare “before” and “after” signature to discern differentiation3.051J/20.340J 6 DNA microarrays: test for gene expression (first commercialized by Affymetrix) array of “probes” comprising surface-bound gene fragments (~20 bases/fragment) Method 1. cells lysed to retrieve mRNA 2. mRNA transcribed to cDNA (complement), which is then transcribed to cRNA 3. cRNA is cut into 35-200 base fragments & labeled with biotin (vitamin H)—referred to as “biotinylation” S CH2CH2CH2CH2 COOH HHOHN NHBiotin has strong binding affinity for streptavidin Ka ~ 1015 L/mol ~50 µm2 probe area = 10M DNA fragments - 2M probes/chip - up to 40,000 genes/chip3.051J/20.340J 7 4. cRNA “cocktail” is added to array, hybridizes with DNA fragments 5. surface-bound cRNA-biotin is labeled with fluorophore-labeled avidin 6. fluorescence spatial readout determines genes being expressed randomized array minimizes error from localized damage Avidin has 4 binding pockets for biotinC CC C C CC C 8 3.051J/20.340J DNA Array Fabrication Lithographic masks used to build up A-C-T-G sequences base-by-base C C C C C C C C C C C C C C C C C C C C A A A A A A A A A A A A A A A A C T A A C S.P.A. Fodor et al., Science 251, 767 (1991).93.051J/20.340J e. Secretion Enzyme-linked immunosorbent assay (ELISA): tests for a particular protein’s presence in cell secretions 1. cell secretions aspirated from culture medium MAb surface made by direct protein adsorption followed by MAb Y YYY YYY Y Y Y YYY YYY YYadsorption or 2. surface coated with soln of monoclonal antibody (MAb) for protein of interest—protein binds MAb if present 3. 2nd antibody (enzyme-linked) is added—binds to protein on surface to create a “sandwich” 4. The enzyme (ex., alkaline phosphatase) catalyzes conversion of a compound to a colored product (amplification) 4. Color intensity is read in spectrophotometer 5. Nonspecific adsorption is determined and subtracted nonspecific adsorption level color intensity cell secretion time10 3.051J/20.340J Alternately, proteins directly adsorbed on a surface may be probed by ELISA in which the second (enzyme-linked) antibody is an antibody to the first