Tips & tricksMicrocell array not sticking to the cell culture ware
Inspect the quality of the insert's seal against the cell cutlure ware by looking through the bottom of the cell culture ware. The presence of air bubbles will reduce the sticking performance of the insert. Remove the insert and try again.
If no air bubbles are present and the insert will not stick to the cell culture ware surface, then remove the insert from the cell culture ware.
Carefully place the bottom surface of the microcell array onto some 3M scotch tape. Note general sticky tape will not work as it can leave glue residue on the insert. The insert can aggresively collect dust/oils that reduces its ability to self-stick to a surface. The scotch tape removes the dust/oils enabling the insert to stick to the culture ware.
Using a pair of tweezers carefully peel the insert off the tape and place it onto a clean section of tape.
Repeat step 3 at least 3 times.
Return the microcell array to the cell culture ware. Make sure that the insert is only ever applied to a dry surface.
Clean the insert and glass surface of the cell culture ware in ethanol and dry with compressed air or nitrogen. Note the inserts will only bond with a glass surface.
Activate the surfaces of the insert and glass cell culture ware using an Oxygen plasma or corona discharge for approximately 15 seconds.
Carefully place the insert onto the glass surface and press down on all areas of the insert making sure it is in contact with the glass. Note if you have made a mistake or a bubble has become trapped between the insert and glass surface you only have one to two minutes to remove the insert and try again before it bonds with the glass.
Hold the insert in place for 30 seconds with pressure from your finger or thumb.
Wait at least 1 hour before starting the ethanol rinse (step 3 in the protocol for using microcell arrays).
John Markham from the National ICT Australia (NICTA) has developed a MATLAB based tool box called MATS (Microgrid Array Tools) to process time lapse microscopy experiments using microgrid arrays. Unlike other processing software MATS has some specific features for use with microgrid arrays like;1.
Automatic recognition of microgrid boudaries. There are three algorithms which can be used with transmission images to find the microgrid boundaries.
Support for spectral unmixing based on the Zeiss Colibri illumination with a quad-band filter block.
Support for automatic position setting. Setting positions that cover all of the 1,000's of wells in a microgrid array is a painful task, so a script is included that automates it, including doing interpolation of the focus to account for a non-level holder.
Support for measurement of FUCCI cell cycle reporter proteins.
Support for multi-treading and GPU acceleration, which allows you to use as many cores on your local machine as the MATLAB parallel tool box will support.
Generation of position specific background images. MATS uses all the images in a stack to try and find the background. THis inly works if the cells are motile and take up only a small proportion of the image.
Support for image registration to account for misalignment at successive time points.