Frequently Asked Questions on Performing the CFU Assay

The in vitro colony-forming unit (CFU) assay is used to evaluate the functionality of hematopoietic stem and progenitor cells (HSPCs) and has been shown to correlate well with in vivo engraftment models. Here, Dr. Jackie Damen (Senior Scientific Advisor, STEMCELL Technologies) answers frequently asked questions (FAQs) about performing the hematopoietic CFU assay.

Explore answers to FAQs on the following topics related to the CFU assay: Reagent Preparation Cell Sample Preparation Setup and Culture Colony Identification and Enumeration

Reagent Preparation

Which MethoCult™ media formulations should I use?

Why should MethoCult™ methylcellulose-based media be thawed at room temperature or in the refrigerator instead of at 37°C?

How do I prepare MethoCult™ methylcellulose-based media for use?

My MethoCult™ medium appears yellow or violet in color after thawing. Can I still use it?

Is it necessary to add antibiotics to the media?

Why are low adherence dishes so important for the CFU assay?

How do I prepare aliquots of complete MethoCult™ media for setting up triplicate or duplicate cultures?

Cell Sample Preparation

Why is RBC depletion important before setting up a CFU assay?

Is trypan blue the most commonly used method for distinguishing live/dead cells, or would you recommend other stains?

Setup and Culture

How many cells should I use to initiate a CFU culture?

Can I use serological pipettes for seeding?

Why does the MethoCult™ medium not cover the entire surface of the well?

How long should I wait after vortexing before plating MethoCult™ tubes with cells added?

Why does the MethoCult™ medium appear to be runny and the colonies are floating or smearing?

Is there anything I can do if my cultures appear contaminated?

Why are my cultures drying out? How can I tell if my cultures are dehydrated?

Colony Identification and Enumeration

Why do I see uneven colony counts in triplicates of the same sample?

Is the scoring of CFU assays subjective?

I get motion sickness counting the colonies. How can I alleviate this problem?

How can I learn to count CFU numbers accurately and reproducibly?

Is it possible to characterize the subsets of the granulocytic lineage differentiated by CFU assay?

Can I leave the human CFU cultures for longer than 14 days?

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Related Resources

CFUs against an orange background

Introduction to STEMvision™ for Automated CFU Scoring Course

Learn how to standardize and automate the scoring of the CFU assay using STEMvision™ in this self-paced digital course. You’ll gain access to lectures, step-by-step protocol videos, and a library of curated resources.

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Wallchart: Human Hematopoietic Progenitors

Hang this wallchart on your lab wall as a handy guide when identifying colonies derived from human hematopoietic progenitors.

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Video: Procedure for Setting Up the CFU Assay

Watch this video for step-by-step instructions on how to set up the hematopoietic colony forming unit (CFU) assay using methylcellulose-based MethoCult™ medium.

Watch Now >