LTC-IC

LTC-IC

-coat 96-well plates with o.1% gelatin/PBS

-preplate 96-well plates with MS5 stromal cells

-sort (transduced) CD34+ cells onto MS5. Use 20 wells per group, plate in limiting dilution from 6, 18, 54, 162, 486, 1458 cells per well

-demidepopulate weekly

-after 5 weeks, remove medium, add 50 ul CFCmix

-score pos/neg wells two weeks later

-use L-Calc to determine LTC-IC frequencies

-plot on y-axis: percentage of negative wells (log scale) on x-axis cells plated per well