How to Set Up Hematopoietic Colony-Forming Unit (CFU) Assays

Thaw the methylcellulose-based medium overnight at 2-8°C or at room temperature for several hours; never thaw at 37°C. Once thawed, shake the bottle vigorously to mix, then let it stand for 20-30 minutes until bubbles rise and the medium becomes transparent.

Using a syringe with a 16-gauge blunt needle, carefully aliquot methylcellulose medium into tubes. Do not use serological pipettes as the medium adheres to surfaces and will not dispense accurately. Store aliquoted tubes in the freezer at -20°C or refrigerator at 2-8°C until use.

Prepare cells according to institutional guidelines and remove red blood cells by lysis with ammonium chloride, sedimentation with Hexa, or immunomagnetic selection. Count cells using a hemocytometer or automated cell counter, then recommend plating at two different concentrations to ensure optimal colony visibility.

Dilute processed cells in IMDM with 2% FBS to ten times the final desired concentration. Add the diluted cells to methylcellulose medium (e.g., 0.3 mL cell suspension to 3 mL medium), vortex to mix, and let stand for 5 minutes to allow bubbles to rise.

Using a syringe with a 16-gauge blunt needle, prime the syringe by drawing up approximately 0.5 mL of the medium-cell mixture and expelling it back into the tube to remove large air bubbles. Then draw medium up to the 2 mL mark on the syringe.

Dispense 1.1 mL of the medium-cell mixture into each 35 mm dish or SmartDish well. Swirl or tilt each dish to evenly distribute the medium and cells across the entire bottom surface.

Surround the plated dishes with sterile water-filled plates to maintain humidity, or fill the interval spaces of SmartDishes with sterile water. Place dishes in an outer container and then into a water-jacketed incubator set to 37°C and 5% CO₂ with a full pan of sterile water at the bottom.

Allow progenitor cells to proliferate and generate colonies of different blood cell types over 7-14 days in the incubator at 37°C and 5% CO₂. Detailed procedures for counting and analyzing colonies are available in technical manuals.