This video provides a step-by-step procedure for setting up hematopoietic colony-forming unit (CFU) assays using semi-solid methylcellulose-based MethoCult™ medium. This procedure includes preparing materials and cells, plating in MethoCult™ medium, and incubating.
This is a five-part instructional video outlining the protocol for setting up and analyzing a matter poetic colony forming unit or cfu assays with meza cellulose-based ethical medium the day before an experiment medium should be thought overnight at two to eight degrees if it is necessary to thaw medium more quickly it may be placed at room temperature to thaw over
Several hours ethical medium should never be thought at 37 degrees once thawed shake the bottle vigorously to mix the medium will appear opaque after shaking let the bottle stand until all bubbles have risen to the top and the medium is transparent again this typically takes 20 to 30 minutes bottles of meth occult medium must be allocated into tubes before use
A lacroix ting and dispensing of meth occult medium should always be done using a syringe and a 16 gauge blunt and needle using a serological pipet or other instrument is not recommended as the medium adheres to the inner surface and the intended volume will not be accurately dispensed if not used immediately al aquatic tubes of meth occult can be stored in the
Freezer at minus 20 degrees until the expiry date on the label or in the refrigerator at two to eight degrees for up to one month prepare cells according to your institutional guidelines cfu assays are best performed using samples with the red blood cells removed red blood cell removal is required for cfu assays that will be counted automatically using stem vision
Red blood cells and culture obscure the visualization of smaller and more diffused colonies in human samples red blood cells may be removed by lysis with ammonium chloride sedimentation with head except or immuno magnetically from cord blood using urethra clear designed specifically for processing up to 16 small volume cord blood samples at one time rbc’s and mouse
Or rat samples may be removed with ammonium chloride after processing cell should be counted with the hemocytometer or automated cell counter plated cell concentrations depend on the donor characteristics processing methods and tissue source it is recommended to place cells at two different concentrations to ensure that an optimal number of colonies will be visible
In at least one set of replicates assays that contain either too few or too many colonies will prevent accurate counting and compromise the accuracy of the measured frequency of progenitor cells in the sample processed and counted cells should be diluted in i mdm with 2% fbs to 10 times the final concentration required in each assay well or dish for example if 1
Times 10 to the power of 4 cells per well are desired prepare a cell suspension at 1 times 10 to the power of 5 cells per mil of which 0.3 mils will then be added to 3 mils of meth occult medium vortex to mix let the tube stand for 5 minutes to allow bubbles to rise to the top prepare a syringe and 16 gauge blunted needle for plating by drying up approximately 0.5
Mil of medium and cells and then expelling it back into the same to this prime’s the syringe and removes large air bubbles at the rubber end of the plunger draw ethical into the syringe up to the 2 point for milk then dispense 1 point 1 mil into each 35 millimeter dish or smart dish well swirl or tilt the 35 millimeter dish or smart dish to evenly distribute the
Medium in cells across the entire bottom surface ensure proper humidity is maintained in the cultures by surrounding the 35 millimeter dishes with plates containing sterile water or filling the interval spaces of a smart dish with sterile water dispose of any remaining media and cells place the 35 millimeter dishes or smart dish into an outer container to further
Ensure the maintenance of adequate humidity then place the entire container of dishes into a water jacketed incubator with a full pan of sterile water at the bottom ensure that the incubator is set to 37 degrees and 5% carbon dioxide individual progenitor cells in these assays will proliferate and generate colonies of different types of blood cells over seven to
14 days for detailed procedures please review the technical manuals for human or mouse colony assays on our website for more information about methe occults please visit mythical calm or email us at info at stem-cell calm
Transcribed from video
How to Set Up Hematopoietic Colony-Forming Unit (CFU) Assays By STEMCELL Technologies
