Frequently Asked Questions: Services
Progenitor (CFC) Assays FAQs
How well can your in vitro assays predict clinical compound-induced hematotoxicity/myelosuppression?
When performed by knowledgeable and experienced scientists such as at ReachBio Research Labs, CFC assays can be very predictive of compound-induced myelosuppression. When a compound causes lineage-specific or multi-lineage hematotoxicity, very often it is the rapidly dividing progenitor cell population that is being affected rather than the mature blood cells themselves. This makes in vitro progenitor (CFC) assays perfectly suited to revealing the effects of compounds on the various blood cell lineages. To see an example of how well the results of in vitro CFU-GM assays can correlate with clinical neutropenia, please visit our HemoRANK-TKI page.
What kinds of compounds can be tested in your predictive hematotoxicity assays?
Almost any compound type can be tested in our in vitro CFC assays. We’ve helped clients working with a wide variety of different compound types, from small molecules to large, complex biologics. Some of the compound types we most often work with are discussed on our Compound Classes page, but if the compound type you’re working on isn’t listed there, chances are that our assay systems will still applicable.
Why do you use functional progenitor cell (CFC) assays instead of just a proliferation assay?
Colony-Forming Cell assays give information on both differentiation and proliferation potential of a given progenitor. Being able to visualize the colony formed by each progenitor verifies that the effects seen are indeed lineage specific. Simple proliferation assays in liquid media cannot give the same detail of information. Even though a cytokine cocktail may be designed to support the growth of a particular progenitor type, other cells will still grow and the proliferation of more abundant cell types can mask the effect a compound has on a relatively rare progenitor cell.
Has the Colony-Forming Cell (CFC) assay been validated as a means to predict compound-induced hematotoxicity?
Yes. ECVAM has validated the human CFU-GM assay for a wide variety of compound classes. More recently, they have extended their studies to evaluate the CFU-MK assay as an in vitro predictor of thrombocytopenia.
How much compound do you need to test and what format should we provide it to you in?
Based on the study proposal, the amount of compound required may vary. In general, though, 2-5mg is sufficient for most in vitro studies. We are happy to accept the compound in powder format or at a stock concentration 1000x higher than the highest desired test concentration. Stock solutions may be made up in DMSO or another solvent of your choice.
How fast is your turn-around time once we provide you with our compounds?
Our turn-around time is based upon the scope of the experiment. In general, though, for in vitro studies we provide an initial data set approximately 16 days after the initiation of the study (CFC assays take up to 14 days before read-out). We strive to initiate your study as soon as we receive your compounds, assuming all the paperwork has been complete. Final reports are generally available 2 weeks following study completion.
How many compounds can you test at once?
We can accommodate a very wide range of study sizes. Depending upon the number of concentrations desired to be tested, we can perform studies with up to 100 compounds at a time.
How many concentrations do you suggest we test?
If you require a very specific IC50 value for a small number of compounds, we would suggest 8-10 concentrations be tested per compound. If, however, you wish to screen and rank a large series of compounds, then 5 concentrations is adequate.
What cell types do you use in your CFC assays?
We most typically use normal human bone marrow mononuclear cells in our CFC assays. However, we can also use MNCs from cord blood and also selected CD34+ cells from bone marrow or cord blood.
We can also test bone marrow and peripheral blood from diseased patient samples.
We also routinely test bone marrow MNCs from mouse, rat, canine and NHP.
What species can you test our compound(s) on?
We can test progenitor cells from human, mouse, rat, canine and NHP. If you require testing of cells from other species, please CONTACT US to discuss your needs. Please be aware that some of the cytokines required to support the various progenitors in CFC assays can be exquisitely species-specific, so not all species may be able to be accommodated.
Can you perform molecular analyses on cells derived from the colonies after the CFC assay has been done?
Yes. We can pluck the colonies from the methylcellulose matrix and perform a variety of downstream tests on the cells. We have the expertise to perform a number of molecular biology techniques in house, but can also send the cells to a third party vendor of your choice if you prefer. Depending on the application, molecular analyses can be performed on cells from individual colonies or on cells from pooled colonies.
Flow Cytometry Services FAQs
Can you perform multi-color flow analysis? If so, how many colors?
Yes. We can perform flow cytometric analyses using up to16 colors using a BD LSR-2 flow cytometer. For studies requiring small number of colors, we generally use other equipment including a Cytek DxP or a BD FACSCanto-2. For studies requiring a very large number of parameters to be assessed at the same time, we would generally employ mass cytometry, utilizing a DVS CyTOF analyzer. This platform allows for up to 30 different parameters to be tested on a single cell and requires SPADE analysis.
Can you analyze flow data for us?
Yes. In addition to performing the flow cytometry assays, we can also analyze the data for you using FlowJo software for studies utilizing small numbers of colors or Kaluza software for studies utilizing higher numbers of colors. We can then work with you to provide the data in a format suitable to your needs. For very high parameter mass cytometry experiments, we typically perform SPADE analysis using the CytoBank platform.
What species can you perform flow cytometry on?
We can perform flow cytometry analyses on a wide variety of species. We routinely analyze cells from human, mouse, rat and NHP.
What is your flow cytometry throughput capability?
We can accommodate a wide range of flow cytometry study sizes, from single tubes to multiple 96-wells. Our multi-parametric capabilities allow for even very complex analyses to be condensed into a reasonably sized study. This is especially important with experiments utilizing primary cells, where the cell number is often limiting.