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Paper text:
we used primary cultures of human cervical epithelial cells prepared from tissues collected from transformation zone of cervix from three cancer patients and three healthy individuals .
the cell isolation was performed by a two - stage enzymatic digestion using dispase to remove the epithelium and then trypsin to disperse the individual epithelial cell27 .
normal and cancerous cell cultures were maintained in keratinocyte serum free medium ( ksfm , invitrogen , carlsbad , ca ) under the same experimental conditions .
ksf - m is a well defined and widely used medium for growth of epithelial cells .
the epithelial cells adhered tightly to the bottom of the plastic dish , and the 60 mm cell culture dishes were mounted on the chuck of the afm with a double sticky tape .
cells were grown to approximately 10% of confluency before using them for scanning ( to avoid induction of squamous differentiation of normal cells ) .
all scanning and measurements related to rigidity were performed on proliferating viable cells maintained to room temperature in hank s balanced salt solution ( hbss ) within 23 hours after removal of growth medium . a nanoscope dimension 3100 ( digital instruments / veeco , inc .
, santa barbara , ca ) atomic force microscope ( afm ) was used in the present study . a standard cantilever holder for operation in liquids
the force - volume mode of operation was utilized . for details , see methods in si text .
louis , mo , usa ) to study glycoconjugates of the pericellular brushes of normal and cancerous cells . using the critical electrolyte concentration method28
, one can also distinguish weakly and strongly charged proteoglycan groups in the cell glycocalix29 . for details , see methods in si text . a confocal microscope ( nikon c1 , 10mw 488 nm and 514 nm argon laser , 100x 1.4 na cfi vc oil immersed objective ) was used .
the cells were attached to a labtek slide ( cell suspension in growth medium was placed to the slide for a couple of days ) .
to visualize the cell and cytoplasmic membrane corrugations , 20 l of 5% ethanol aqueous solution of nile red fluorescent dye was added to 1ml of buffer . within 15 minutes after placing the cells in this buffer , viable cells were imaged directly in that buffer .
cells were fixed with karnovsky'sfixative at 4c for 2 days , post - stained with 1%osmium tetroxide , further stained with 0.67% aqueous ruthenium tetroxide .
sem imaging was done on freeze - dried samples . for tem - type of imaging
, cells were further dehydrated with absolute ethanol and embedded in spurr epoxy ( firm formulation ) . thinsections ( 2050 nm ) were viewed using electron back - scattering sem mode .
the data have been fitted to the analytical expression using the levenberg - marquardt and simplex algorithms .
the error of the fit is one standard deviation . when calculating  error - bars  for average values , individual standard deviations are discarded as small , and rms value of mean values is calculated .
so , the  error - bars  for average values should be called  variability bar  .
we used primary cultures of human cervical epithelial cells prepared from tissues collected from transformation zone of cervix from three cancer patients and three healthy individuals .
the cell isolation was performed by a two - stage enzymatic digestion using dispase to remove the epithelium and then trypsin to disperse the individual epithelial cell27 .
normal and cancerous cell cultures were maintained in keratinocyte serum free medium ( ksfm , invitrogen , carlsbad , ca ) under the same experimental conditions .
ksf - m is a well defined and widely used medium for growth of epithelial cells .
the epithelial cells adhered tightly to the bottom of the plastic dish , and the 60 mm cell culture dishes were mounted on the chuck of the afm with a double sticky tape .
cells were grown to approximately 10% of confluency before using them for scanning ( to avoid induction of squamous differentiation of normal cells ) .
all scanning and measurements related to rigidity were performed on proliferating viable cells maintained to room temperature in hank s balanced salt solution ( hbss ) within 23 hours after removal of growth medium .
a nanoscope dimension 3100 ( digital instruments / veeco , inc . , santa barbara , ca ) atomic force microscope ( afm ) was used in the present study . a standard cantilever holder for operation in liquids
the force - volume mode of operation was utilized . for details , see methods in si text .
louis , mo , usa ) to study glycoconjugates of the pericellular brushes of normal and cancerous cells . using the critical electrolyte concentration method28
, one can also distinguish weakly and strongly charged proteoglycan groups in the cell glycocalix29 . for details , see methods in si text .
a confocal microscope ( nikon c1 , 10mw 488 nm and 514 nm argon laser , 100x 1.4 na cfi vc oil immersed objective ) was used .
the cells were attached to a labtek slide ( cell suspension in growth medium was placed to the slide for a couple of days ) .
to visualize the cell and cytoplasmic membrane corrugations , 20 l of 5% ethanol aqueous solution of nile red fluorescent dye was added to 1ml of buffer . within 15 minutes after placing the cells in this buffer , viable cells were imaged directly in that buffer .
cells were fixed with karnovsky'sfixative at 4c for 2 days , post - stained with 1%osmium tetroxide , further stained with 0.67% aqueous ruthenium tetroxide .
sem imaging was done on freeze - dried samples . for tem - type of imaging
, cells were further dehydrated with absolute ethanol and embedded in spurr epoxy ( firm formulation ) .
the data have been fitted to the analytical expression using the levenberg - marquardt and simplex algorithms .
the error of the fit is one standard deviation . when calculating  error - bars  for average values , individual standard deviations are discarded as small , and rms value of mean values is calculated .
so , the  error - bars  for average values should be called  variability bar  .
the atomic force microscope is broadly used to study the morphology of cells15 but it can also probe the mechanics of cells . it is now known that cancerous cells may have different mechanical properties than normal cells68 but the reasons for these differences are poorly understood9 . here 
 we report quantitatively the differences between normal and cancerous human cervical epithelial cells by considering the brush layer on the cell surface . 
 these brush layers , which consist mostly of microvilli , microridges , and cilia are important for interacting with the environment . 
 deformation force curves obtained from cells in vitro are processed according to the ' brush on soft cell model 10 . 
 we found that normal cells have brushes with one length while cancerous cells displayed long and short brushes with significantly different densities . 
 the observed differences suggest that brush layers should be taken into account when characterizing the cell surface by mechanical means .