Cell array (CA) production

3.5.1   Functionalization  of  chambered  cover  glasses    

LabTek  chambered  cover  glasses  (Nalge  Nunc  International)  were  treated  with   1M  NaOH  (J.T.  Baker)  for  15  min  and  washed  three  times  with  H2O  afterwards.    

3.5.2   Preparation  of  transfection  mix  plate    

The   array   production   was   based   on   the   work   of   (Erfle   et   al.,   2007).   The   production   of   transfection   mixtures   were   handled   using   an   automated   MICROLAB   STAR   Line   robot   system   (Hamilton   Robotics   GmbH).   For   each   spot   mixture  assembled  on  a  384  well  plate,  3  μl  Sucrose/OptiMEM  (Sigma-­‐Aldrich  /   GIBCO)  was  mixed  with  3.5  μl  Lipofectamine  2000  (Invitrogen).  For  cDNA  arrays,  

we  used  1  µg  plasmid  cDNA  per  spot  mixture  for  single  reverse  transfections  or   2x  0.5  µg  for  co-­‐transfections  with  two  plasmids.  1  µg  plasmid  cDNA  was  filled   up  with  H2O  to  reach  a  5  µl  total  volume  (0.20  µg/µl)  and  used  for  a  spot  mixture.  

For  siRNA  arrays,  lyophilized  siRNA  (4nmol)  were  solved  in  200  μl  siRNA  (1x)   buffer   (Thermo   Scientific)   to   generate   a   20   μM   concentration.   For   each   spot   mixture,  we  used  2.5  µM  siRNA  in  5  µl  volume.  The  5  µl  siRNA/cDNA  solutions   were  mixed  with  the  Sucrose/Lipofectamine  on  the  384  well  plate  and  incubated   for   20   min.   7.25   μl   of   0.2   %   Gelantin/H2O   (Sigma-­‐Aldrich)   supplemented   with  

0.01   %   bovine   fibronectin   (Sigma-­‐Aldrich)   was   added   to   each   well   after   incubation.   On   cDNA   spots   we   have   used   a   final   concentration   of   0.05   %   fibronectin  what  had  increased  the  reverse  transfection  efficiency.    

3.5.3   Contact  spotting  routine    

Contact   spotting   was   performed   with   a   QArray2   robot   (Genetix).   The   384   well   source   plate   and   functionalized   LabTek   chambers   were   placed   on   the   spotter   table   and   immobilized   by   the   vacuum   system.   The   humidity   and   temperature   was  set  to  60  %  and  20  °C  during  the  spotting  performance,  respectively.  Four   solid  microarray  steel  pins  with  a  tip  diameter  of  500  μm  (Array-­‐it  corporation)   were   placed   in   horizontal   position   in   the   spotter   head   (front   row,   first   four   positions   from   left)   (Figure   3.3   a).   The   source   order   was   set   to   columns.   The   slide  design  was  set  to  print  grids  of  32  rows  and  12  columns  with  a  1125  μm   square  unit  cell  (distance  between  the  center  of  neighboring  spots).  This  yields   384  samples  per  LabTek  chamber.  Number  of  stamps  per  ink  and  spot  was  set  to   3.  The  inking  time  (time  that  the  pins  stays  in  the  384  well  plate)  and  the  stamp   time  (time  that  the  pin  is  in  contact  with  the  glass  of  the  LabTek  chamber)  was   set  to  110  ms.  The  print  depth  was  set  to  180  μm.    The  multi-­‐stamp  timing  was   adjusted  to  immediate  (the  stamps  will  be  done  consecutively).  After  every  spot   stamping  a  washing  step  with  the  following  protocol  was  performed:  Wash  3  sec.   with  H2O,  wash  3  sec.  with  EtOH,  wash  1  sec.  with  H2O  and  dry  5  sec.  by  air  flow.  

A   csv   sheet   was   prepared   containing   all   positions   of   wells   on   the   source   plate   and  name  of  the  siRNA/cDNA  samples  that  each  of  them  contains.  The  file  was   imported  into  the  QSoft  Data  Tracking  software  (Qaray2  system)  to  generate  a   galfile  that  was  used  for  the  automated  microscopy  system.  The  routine  was  set   to  “normal  run”  to  print  the  array  by  using  all  wells  of  the  384  well  source  plate.    

3.5.4   Spotting  of  reference  points    

After   finishing   the   384   spot   routine,   6   ink   spots   were   spotted   on   top   of   the   generated   array.   These   ink   spots   are   used   later   as   given   coordinates   of   the   distinct  array  grid  to  define  the  positions  of  all  other  spots  on  the  array.    To  keep   the  same  array  grid,  the  vacuum  system  to  immobilize  array  chambers  was  not   interrupted   when   changing   to   the   ink   spotting   routine.   Approx.   15   µl   super-­‐   stay24color®  #450  fire  garnet  (Maybelline  New  York)  was  placed  on  position  A1  

on  a  new  source  384  well  plate.  We  found  out  that  this  trademark  was  not  toxic   to   cells   when   present   on   array   surfaces   and   was   highly   water-­‐proof,   thereby   providing  optimal  conditions.  The  four  500  μm  diameter  pins  were  removed  and   one   300   μm   diameter   pin   was   placed   on   the   robot   head   (front   row,   fourth   position   from   left).   The   slide   design   was   set   to   print   grids   of   32   rows   and   12   columns  like  described  previously,  but  only  the  first  position  of  every  4x4  block   was  set  for  spotting.  The  routine  was  set  to  “test  run”  what  means  that  the  inking   position  on  the  source  384  well  plate  is  always  A1.  By  spotting  with  one  pin,  six   distinct  ink  spots  on  array  position  1A,  5A,  9A,  13A,  17A  and  21A  were  printed.   Number  of  stamps  per  ink  and  spot  was  changed  to  1.  The  inking  time  and  the   stamp  time  was  set  to  10  ms.  The  washing  step  was  inactivated  and  the  pin  was   cleaned   with   acetone   by   hand   after   every   eighth   arrays.   After   spotting,   arrays   were  dried  for  12  hours  (or  stored  longer)  in  a  box  containing  silica  Gel  Orange   pearls  (Carl  Roth  GmbH).  Reference  spot  are  shown  in  (Figure  3.3  a).  

3.5.5   Reverse  transfection  on  CA      

Spotted   arrays   were   warmed   for   10   min   at   37   °C   in   the   cell   culture   incubator   before  cell  seeding.  Cultured  HeLa,  A431,  MCF7  or  stable  transfected  EGFR-­‐GFP   MCF7   cells   were   washed   with   DPBS   (PAN   Biotech   GmbH),   trypsinized   (PAN   Biotech  GmbH)  and  counted  by  using  a  cell  counter  system  (Beckman).  2.50.000   cells   per   array   for   siRNA   CA   or   300.000   cells   per   array   for   cDNA   CA   were   supplemented  in  4  ml  DMEM  (PAN  Biotech  GmbH)  complete  (+P/S)  and  seeded.   In  case  of  HeLa  cells,  arrays  were  washed  three  times  with  DMEM  after  20  min   after   seeding.     Arrays   were   incubated   for   24   hours   for   reverse   transfection   of   cDNAs  or  48  hours  for  reverse  transfection  of  siRNAs.  Cells  were  incubated  at  37   °C  and  5  %  CO2.