CELL ADHESION

 

Procedure:

 

1) Dissolve/dilute coating substrate in ddH2O at 4C.  A common working dilution for the laminin-1 positive control and BSA (Sigma A8412) negative control is 40 g/ml.  For SN-peptide, use 20 M (MW of SN-peptide is 2412) for plateau or 2.5 - 5 M for half-maximal adhesion.  For fragment E8 or laminin-1 plateau and half-maximal adhesion are usually achieved at 0.5 and 0.05 M, respectively.  Note: SN-peptide should be used right after dissolution in ddH2O.  Activity decreases with storage, even frozen.  A reliable approach is to weigh out 100 g aliquots with a microbalance for use with individual experiments.

 

 2) Add coating solution (100 l/well) with multipipetter to wells of a 96 well tissue culture plate (Costar #3595), cover, and place at 4C overnight.  Coat in triplicate or quadruplicate.

 

3) Invert plate and shake out coating solution.  Pull off remaining coating solution from each well with a yellow tip pipetter.

 

4)  Dilute 7.5% BSA (Sigma A8412) to 1% in ddH2O.  Add 100 l/well with multipipetter, cover and place at 4C for 4 hrs.  Thaw 10 x trypsin/EDTA (then dilute to 1 x in PBS), warm PBS and serum-free medium.

 

5) In last 45 min of block, pull off medium from cells in T75 flask, and add serum-free medium.  Replace in incubator for  30 min.  Subsequently, pull off medium, wash with PBS, add 1 x trypsin/EDTA for 1 - 3 min, pull off released cells, wash flask with 20 ml of serum-free medium, pellet cells in 40 ml of serum-free medium, make up in 6 ml of serum-free medium and count (15 l of suspended cells plus 15 l of trypan blue; add 15 l to each side of hemocytometer; cell#/ml = combined count from both sides x 104).  Dilute cells to 2.0 x 105/ml in serum-free medium. 

 

6) Invert plate and shake out BSA blocking solution.  Pull off remaining blocking solution from each well with a yellow tip pipetter.

 

7) Pour cells in Reagent Reservoir (Costar # 4870), rock to suspend, remove 100 l/well with multipipetter and add to wells.  Repeat rock/resuspension prior to removing cell suspension for each row.  Place in incubator for 30 - 60 min (37C).

8) Examine plate in invert microscope.  Photograph selected wells if desired.  Invert plate gently onto an absorbent diaper pad.  Pull off remaining cell solution from each well with pipetter.

 

9) With multipipetter, slowly add 100 l/well of serum-free medium down the side of each well (tilt plate; PBS is not recommended for this wash).  Invert plate gently onto an absorbent diaper pad.  Pull off remaining wash solution from each well with pipetter.

 

10) Slowly add 100 l/well of serum-free medium down the side of each well.    Examine plate in invert microscope.  Cells in BSA negative control wells should be rare (if not, repeat wash).  Adherent cells in SN-peptide wells remain mainly rounded or slightly spread.  An exception is M2 melanoma, which spreads rapidly on SN-peptide.  Adherent cells in laminin-1 wells should be all spread.  Invert plate gently onto an absorbent diaper pad.  Pull off remaining wash solution from each well with pipetter.

 

11) With multipipetter, slowly add 100 l/well of freshly diluted 1% glutaraldehyde in PBS.  Fix for 10 min at room temp.  Invert plate gently onto an absorbent diaper pad.  Pull off remaining fix solution from each well with pipetter.

 

12) With multipipetter, add 100 l/well of freshly filtered (use 0.2 m syringe filter) crystal  violet (0.1% in ddH2O; Serva # 27335).  Stain for 25 min at room temp.  Invert plate onto an absorbent diaper pad, then wash plate gently by immersion in a plastic tray containing tap water.  Invert plate  onto an absorbent diaper pad.  Pull off remaining wash from each well with pipetter.  Reimmerse in fresh tap water.  Invert plate  onto an absorbent diaper pad.  Pull off remaining wash from each well with pipetter.  Repeat an additional time if required.  Allow to dry for 5-10 min at room temp.

 

13) With multipipetter, add 50 l/well of 0.5% Triton X-100 (diluted in ddH2O).  Allow to solubilize overnight at room temp. in a drawer.  Read at OD 595.  BSA background should be less than 0.1 OD.  Laminin value should be about 1.0 OD.  Plateau SN-peptide value is usually 70-80% of laminin.