Importance: wound healing immune host defense cancer therapy tissue engineering bioprocessing/synthesis of theraputic glycoproteins

Transcription

1 1 Lecture 14: Quantifying ell Function c. ell Proliferation ssays Importance: wound healing immune host defense cancer therapy tissue engineering bioprocessing/synthesis of theraputic glycoproteins ell number 1. count via microscopic observation or oulter counter 2. compute specific cell proliferation rate const: 1 dn d (ln N ) k g = = (units: t -1 ) N dt dt N = # of cells at time t ell 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 DN synthesis (~18-72 h) S G 1 S = DN synthesis (~6-8 h) G 2 = gap between DN synthesis & mitosis (~2-3 h) G o = quiescent cells

2 2 tritiated-thymidine uptake 1. cells exposed to [ 3 H]thymidine pulse which labels S-phase cells only 2. % S obtained from autoradiography (g 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 G1 + t S + t G2 tcycle t M 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 % labeled mitoses t S t G2 time H] thymidine pulse [ 3 *autoradiography is also used with PGE for protein identification

3 3 flow cytometry* 1. cells DN labeled with DN-specific fluorescent dye (ex. acriflavine, ethidium bromide) 2. count cell # vs. fluorescence using laser excitation 3. fluorescence intensity ~ DN present G 1 :S:G 2 :M DN (& fluorescence) ratio is 1:1-2:2:2 G 1 S G 2 +M cell # area % relative DN content 100 dvantages: Drawback: - no radiolabeling - M & G 2 populations lumped - faster than autoradiography *flow cytometry is also used to determine expression of specific cell receptors by labeling with fluorescent antibodies

4 4 d. ell Differentiation ssays Importance: - change in phenotype renders specialization of function - characterized by changes in protein synthesis, genes expressed & secretions 2D Gel Electrophoresis (SDS-PGE): 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 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 ph 2 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 constant

5 5 5. gel is placed under E-field, separating protein chains via molecular weight low molecular weight species move faster µ = µ (c gel, E, MW protein ) SDS-PGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis MW pi 6. compare before and after signature to discern differentiation

6 6 DN microarrays: test for gene expression (first commercialized by ffymetrix) array of probes comprising surface-bound gene fragments (~20 bases/fragment) - 2M probes/chip - up to 40,000 genes/chip ~50 µm 2 probe area = 10M DN fragments Method 1. cells lysed to retrieve mrn 2. mrn transcribed to cdn (complement), which is then transcribed to crn 3. crn is cut into base fragments & labeled with biotin (vitamin H) referred to as biotinylation Biotin has strong binding affinity for streptavidin K a ~ L/mol H HN O S NH H H 2 H 2 H 2 H 2 OOH

7 7 4. crn cocktail is added to array, hybridizes with DN fragments randomized array minimizes error from localized damage 5. surface-bound crn-biotin is labeled with fluorophore-labeled avidin vidin has 4 binding pockets for biotin 6. fluorescence spatial readout determines genes being expressed

8 8 DN rray Fabrication Lithographic masks used to build up --T-G sequences base-by-base T S.P.. Fodor et al., Science 251, 767 (1991).

9 9 e. Secretion Enzyme-linked immunosorbent assay (ELIS): tests for a particular protein s presence in cell secretions 1. cell secretions aspirated from culture medium Mb surface made by direct adsorption or protein adsorption followed by Mb Y YYY YYY YY Y YYY YYY Y Y 4. olor intensity is read in spectrophotometer 2. surface coated with soln of monoclonal antibody (Mb) for protein of interest protein binds Mb if present 3. 2 nd 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) 5. Nonspecific adsorption is determined and subtracted color intensity nonspecific adsorption level cell secretion time

10 10 lternately, proteins directly adsorbed on a surface may be probed by ELIS in which the second (enzyme-linked) antibody is an antibody to the first antibody.