What do you mean by resistant cells? Have they been transfected and selected for expression of a cDNA or shRNA? Have they been grown on plastic for selection? What is the difference to your control cells?

I work with stem cells , I usually seed more cells, so after repeated washing I still have enough cells in the chamber slide.

 Your resistant cells may have become less adherent during their transformation. Try to limit the trypsinization or use accutase for dissociation. You may have to give the cells longer to adhere but I would also recommend seeding the cells on coverslips in a 24-well plate, and then spin down the cells using a plate holder in a swing bucket centrifuge. Additionally you may want to consider using ECM coated coverslips ( ie collagen, fibronectin).  Good Luck.

I faced a similar problem with using low adhering cells in IF, I would recommend coating your coverslips with fibronectin, as Richard suggests. I used bovine fibronectin (Sigma F4759) at 15 ug/m diluted in PBS, coating wells/coverslips with 2.5 ug fibronectin per cm squared. I added the required amount of volume and then let the solution evaporate in a laminar flow hood for 3-4h at room temperature.

By resistant cell lines i mean they are selected for a particular drug (not by transfection) by continous culturing in presence of drug. I could rectify the problem by seeding the cells after trypsininsing in FBS for 2hours followed by normal culture media. this is generally used for seeding extremely sensitive cells.

Thank you all for the suggestions ....