What kind of data will I be able to obtain using the CellDirector assays?
CellDirector microfluidic assays will enable you to collect high quality data on for example cell migration, proliferation, apoptosis, and cell morphological changes indicative of differentiation – in response to defined concentration gradients of the substance of your choice. Cell behavior in response to defined gradients is preferentially recorded using time-lapse microscopy or video microscopy.
Why are cell migration studies so important?
The human body has billion of cells that need to communicate in order to know things like their position in the body and their tasks. This communication is done by cells sending out, and responding to molecular substances in their close environment, and specifically by responding to concentration changes of these substances – concentration gradients.
These processes are very essential and rich targets for intervention in key physiological and pathological phenomena such as cancer metastasis and wound healing. Recent insight from the emerging fields of quantitative and systems biology highlight the importance of analysing individual cells instead of just bulk cultures.
What biological or synthetic molecules can be used for creating the concentration gradient?
Any substance that is water-soluble can be used to generate a gradient. Examples of biomolecular substances include drug candidates, protein growth factors, peptides, hormones, morphogens, metabolites and vitamins.
What kind of biological material can I study using the CellDirector assays?
CellDirector products can be used to study any type of adherent cell line, primary cell type, small cell cluster or tissue explant that can be cultured in vitro.
Do I need to matrix pre-coat CellDirector 2D?
CellDirector 2D is delivered uncoated. It is possible to pre-coat CellDirector 2D according to your protocol of choice, but it is not always necessary. Some cells adhere to the glass surface directly without any pre-coating, for example neutrophils.
Can CellDirector 2D be coated?
Yes, pre-coating of CellDirector 2D is achieved by simply injecting for example your collagen or fibronectin solution prior to loading of the cells.
How can I get my non-adherent cells to adhere?
CellDirector 2D is developed to suit the study of all types of adherent cells. To be able to use it for your non-adherent cells, you need to have them to adhere to the glass surface, before building up a stable gradient and study chemotaxis.
How do I optimise my coating and cell seeding protocol?
There are several things in your protocol that you can optimize to match your cells with the experimental setting. Pre-coating can improve the cells to attach to the surface – both concentration and incubation time matters and should be optimised. As for standard cell culture procedures, typical coatings consist of gelatin, collagen I, fibronectin, poly-D-lysine, and so on.
When you then seed your cells, the time for your cells to adhere can vary, and although 60 min up to 4 hours should be enough, it might be that an overnight incubation suits your cells the best. In that case, make sure you always put a wet tissue in the petri dish together with the CellDirector assay to avoid evaporation during incubation. You may need to further differentiate the cells and or activate them for improved adherence.
If you are not sure what works for your specific cells, try out on glass cover slips before moving ahead with the CellDirector assays.
Is it possible to use the HL-60 cell line?
TheHL- 60 cells may be used after differentiation into neutrophils before seeding the cells into CellDirector 2D. Before having differentiated into neutrophil-like cells, the HL-60 cells are not adherent.
Do I need to matrix pre-coat CellDirector 3D?
There is no pre-coating of CellDirector 3D prior to adding your cells. You add your cells/tissue of interest together with the matrix used in a cell-matrix mixture.
What type of matrix can be used in CellDirector 3D?
All matrices that can be applied in soluble form and which thereafter polymerise can be used in CellDirector 3D, for example matrices based on collagen or Matrigel™.
How do I avoid evapororation when incubating over night?
In case you do the “adhesion of cell” step over night, make sure you add a wet tissue in the petri dish together with the CellDirector assay. This is a recommended procedure for long time incubations to avoid evaporation.
How do I mount CellDirector in my microscope?
CellDirector is ideally mounted into CellDirector Holder (REF 70-001), a metal microscopic holder that has the same outer dimensions as a standard microtiter plate. CellDirector can also be mounted in standard adjustable frames used for small petri dishes.
What equipment is needed to start a CellDirector experiment?
CellDirector experiments require a minimum of equipment in addition to your imaging microscope. A Start-Kit allows you to quickly set up your first experiments, and can be ordered with either CellDirector 2D (Start-Kit 2D, REF 21-001) or CellDirector 3D (Start-Kit 3D, REF 20-001).
Can I control the oxygen and CO2 levels for my cells within CellDirector?
The design and material composition of all CellDirector make the assays completely gas permeable, which means that the gas composition inside CellDirector equals the gas composition outside the assays. To provide your cells continuously with controlled levels of oxygen and CO2, a cell culture incubator is recommended for your microscope. Most time-lapse
microscopes have an incubator solution, so this is normally no problem.
How do I supply my cells with new nutrient over a period of time?
New cell media is supplied to the cells due to the continuous fluid flow of cell media from the syringes in the syringe pump. A CellDirector 2D experiment can last for up to 36 hours. The integrated waste handling absorbs the waste fluid. CellDirector 3D can be run for several days. The waste handling absorbs waste for at least 72 hours.
What concentration of my chemoattractant or chemoreppelent should I start with?
CellDirector assays generate steady-state gradients ranging from your lowest concentrated solution (sink solution) applied, to your highest concentrated solution (source solution) applied. For evaluation, we advise you to start with a serum gradient 0-10% FBS to create directionality, before proceeding to single substances, or cocktails of substances. A serum gradient is also good as a start for comparing knockdowns or other modified cell lines.
Can several “source concentrations” be tested with CellDirector, since cells probably have a minimum concentration initiating chemotaxis?
For each experiment, a single CellDirector is loaded into a CellDirector Holder and placed in your microscope stage. You apply a single source solution with a g/ml of your chemoattractant. The experiment will generate a continuous, steady-state gradient with your source concentration as the highest concentration value.
Analyse the cell migration responses with our Tracking Tool software: use its Selection Tool command and determine the differences in cell chemotaxis within different concentration regions with a single click.
Can the quality and the age of my cells influence the results?
The quality and age of the cells can make a big difference for the migration responses. This is typically the case for bone marrow derived cells, like mast cells, eosinophils and neutrophils. An overall tendency is that young cells migrate to a larger extent than older cells. Remember that there can be differences between batches of cell lines.
How can I visualise the gradient that is formed?
Gradient profiles can be monitored by using real-time fluorescence microscopy. Either label your substance of interest or use Gradient Marker FITC 20K (REF 80-001) or Gradient Marker TRITC 20K (REF 81-001) that are included in Start-Kit 2D and Start-Kit 3D.
The gradient profile of the Gradient Marker products is similar to several soluble growth factors and can thus be considered as a good approximation when working with growth factors such as FGF2, VEGFA and EGF. Please see the product information for the individual Gradient Marker products for more information.
How should I dilute the Gradient Marker products?
Add the Gradient Marker product of choice to your source solution to visualise the gradient formed within CellDirector. A 1:20 dilution is recommended, but higher concentrations might be needed depending on the excitation source of the microscope used.
How can I determine if my gradient molecule of interest interacts with the matrix?
The molecule of interest can be labeled with a fluorescent dye using a standard kit, and this enables a direct readout of gradient formation and dynamics. The affinity of your gradient molecule for the matrix will be reflected by the rate of accumulation in the matrix.
Is CellDirector a disposable product and is it delivered sterile?
Yes. All CellDirector assays have been sterilized by ethylene oxide and are intended for single use only to eliminate the risk for sample contamination.
How many CellDirector assays are included in a box?
One box contains 10 packages of CellDirector. Each package contains a CellDirector assay together with all required single-use syringes and tubes for an experiment.
What type of microscope equipment is required?
The CellDirector assays have excellent light transmission from 300-1200 nm, and cells can be analysed by using a standard upright or inverted time-lapse microscope. We recommend that your microscope is equipped with a motorised x-y stage and a cell culture incubator for best results. Notably, all CellDirector assays are fully compatible with advanced confocal microscopy.