CFU Assay Set Up for Custom Hematopoietic Training Courses

Open the course package and verify all components match the pack list. Store reagents under appropriate conditions: cells in liquid nitrogen at −135°C, and methyl cellulose and IMDM with 2% FBS at −20°C.

One day before the assay, thaw methyl cellulose and IMDM overnight in the refrigerator or thaw at room temperature. On the day of the procedure, disinfect the biosafety cabinet with 70% ethanol or isopropanol and place all reagents and equipment inside.

Remove the cell vial from liquid nitrogen and thaw quickly in a 37°C water bath for approximately 2 minutes until only a small piece of ice remains. Wipe the vial with 70% ethanol, transfer cells to a 15 mL tube, and slowly add 10 mL of IMDM drop-wise while swirling.

Centrifuge the cell suspension at 300× G for 10 minutes. Carefully discard the supernatant and resuspend cells in the remaining medium by flicking the tube bottom until homogeneous and clump-free. Add 2 mL of IMDM to reach the final cell stock volume.

Perform nucleated cell count and viable cell count using either a hemocytometer or validated automated cell counter. Record nucleated cell concentration in 10^6 cells/mL format and calculate percent viability by dividing viable count by total count and multiplying by 100.

Calculate the viable cell concentration and determine volumes of cell stock and IMDM required using Table 1 on the worksheet. Mix the calculated volumes gently to prepare 1 mL of the ten-times plating density solution.

Mix 0.3 mL of ten-times plating density with 3 mL of methyl cellulose medium, vortex for 4 seconds, and let stand for at least 5 minutes. Using a 3 mL syringe with 16-gauge blunt needle, dispense 1.1 mL into each well of duplicate 35 mm dishes, coating the bottom evenly.

Place each 35 mm dish into a 100 mm dish, add a third 100 mm dish containing sterile water with the lid removed to maintain humidity, and cover both dishes. Incubate at 37°C, 5% CO₂, and 95% humidity for 14 days.