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info@stemcell.com . Photomicrographs are taken with an Olympus DP71 camera with a 60x oil objective. Divide these stock solutions into small aliquots and keep at -20 °C to avoid repeated freeze-thaw.Characterize and score the colonies according to their morphology with an inverted microscope at 40x magnification in a culture dish marked with a scoring grid.

Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction.

The slightest bacterial or fungal contamination will overwhelm the culture. See manufacturer's instructions for further colony subclassification.We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood.
Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. The number and morphology of the colonies are documented. Remove the BSA solution, add 0.5 ml of virus preparation to each well, and centrifuge at 2200 x g at 4°C for 15 minutes.Prepare 0.02% EDTA in HBSS and store at 4°C.Vortex vigorously to make the mixture rise fully and fall 3-4 times. Culture for 14 - 17 days.Prepare GaLV-pseudotyped retrovirus encoding the DNA of interest and store in aliquots at -80 °C. This procedure is outside the scope of this article, but is described elsewhere (1-3).Immediately, dip the slide carrier in Wright/Giemsa stock solution (#2) for 5 min.Rinse each well with cold 0.5 ml HBSS (+ Ca & Mg) or IMDM.For further analysis of differentiation and proliferation, cells from the entire CFC assay plate are recovered by suspending in several volumes of room temperature 2% FBS/IMDM. 1. Vortex vigorously to mix and let the tubes stand at least 5 min to let any bubbles present to rise to the surface before adding cells.The entire CFC assay plate may be scanned without magnification using a regular scanner at 600 dpi, and low-power (40x) photomicrographs of colonies may be taken using an inverted microscope equipped with a color camera.Prepare complete IMDM medium with final concentrations of 20% FBS, 2 mM Glutamine, 100 units/ml Penicillin/Streptomycin (PS).

For the purposes of our assay, the colonies are classified into 3 categories: pure erythroid, myelomonocytic, and mixed.

After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days.

Repeat the procedure by adding 2% FBS/IMDM that is the same volume as the diluted cell suspension at 3 min intervals, swirling gently in between additions, until the final volume reaches 32 ml.Since the methylcellulose-based culture is relatively long-term and the CFC medium does not contain antibiotics, it is of paramount importance to prevent contamination of the cell cultures, particularly during and after sorting. This assay is useful for assessing myeloid but not lymphoid differentiation. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Interpretation of flow cytometry and cell morphology requires experience; consultation with trained personnel is recommended. It has the advantage over liquid cultures of being a clonal assay, such that the colonies represent the progeny of a single progenitor and can be individually removed for further analysis. The limitation of the CFC assay is that it is not adequate for the detection of more immature progenitors or hematopoietic stem cells; such cells are detected using the long-term culture-initiating cell (LTC-IC) assay (6, 7). Take 3,000 virus-transduced, sorted cells into a sterile microtube containing cold 2% FBS/IMDM. Methylcellulose-based CFC media are very viscous and special care should be taken while handling them to mix evenly and eliminate bubbles before pouring into plates.

Add the following cytokines just before use: 100 ng/ml FLT-3 ligand, 20 ng/ml GM-CSF, 100 ng/ml SCF, 100 ng/ml TPO, 50 ng/ml IL-3, and 100 ng/ml IL-6.Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. Incubate for 30 minutes at room temperature.Flow cytometry is performed at the High Speed Cell Sorter Core of the Alvin J. Siteman Cancer Center at Washington University School of Medicine on a FACScan flow cytometer upgraded to 5 colors and two lasers (BD Biosciences) and analyzed using CellQuest (BD Biosciences) or FlowJO v7.2.4 (Tree Star, Inc., Ashland, OR, USA) software.Transfer the carrier into Wright/Giemsa buffer, pH 6.4 (#3) and incubate for 10 min.Department of Pathology and Immunology, Washington University School of MedicineRemove the virus solution from wells and repeat the virus loading described in 2.10 three more times.Attach a 16 gauge blunt-end needle to a 3 ml syringe and draw up 2.2 ml.