Q: What size should the tumors be prior to mincing?
A: The size of the tumor used in the assay is not critical because there is a step that adjusts for cell number (step 14 of the Assay Protocol found in the product manual). It is difficult to recommend a size because the cell density will vary, but we used a 160mg lung tumor when developing this assay. If the tumor is large enough to be minced, it will likely have enough cells for the assay.
Q: How does this assay eliminate normal cells from tumor cells?
A: This assay relies on the ability of cells to form colonies in soft agar. Tumors consist of three types of cells: normal cells, cancer cells that cannot form colonies in soft agar, and cancer cells that can form colonies in soft agar. After culturing tumor cells for 6-8 days in soft agar, only the cells that are able to grow in soft agar will form colonies. The colonies are then separated from the other cells based on size using a filter.
Q: Is this kit compatible with any cell type?
A: This kit is not cancer type specific; it can be used for any solid tumor tissue samples.
Q: What enzyme do you recommend for the tumor digestion enzyme listed under "Materials Not Supplied"?
A: The enzyme mixture we used contains 0.05 mg/mL Collagenase I, 0.05 mg/mL Collagenase IV, 0.01 mg/mL DNase I in HBSS.
Q: Can a cell line be established from cells isolated with this kit?
A: This kit isolates clonogenic cells from a solid tumor sample following a 6-8 day incubation in soft agar based on the separation of the colonies that have formed during this incubation. Isolated viable cells can be cultured and a cell line can be established if there are enough viable cells. Although we have not established a cell line using this assay, the customer can expect 50-1000 colonies after a one week incubation.
Q: How likely is it to get a stable cell line?
A: Based on publications, cells from most types of human solid tumors can form colonies in soft agar culture with plating efficiency from 0.01 to 1%. On average, when 500,000 cells from solid tumor are plated, after one week culture, 50-1000 colonies are expected.
Q: Is it possible to quantify isolated cells?
A: This kit does not provide a quantitation step, but isolated viable cells can be cultured or used for subsequent testing, such as by flow cytometry analysis.