Expression and signaling in DT40 cells with immunoglobulin knockouts and chimeric immunoglobulin insertions.

Wild-type cells (DT40 wt), V_L knockout (Igl^KO), V_H knockout (Igh^KO) and V_L-V_H double knockout cells (Igl^KO, Igh^KO) as well as huV_K insertion (Igl^huVK), huV_H insertion (Igh^huVH) and huV_K-huV_H (Igl^huVK, Igh^huVH) double insertion cell lines were analyzed for expression and signaling of immunoglobulin receptors. a) 1×10⁶ cells were lysed and immunoglobulin heavy chain expression determined by Western blotting using goat-anti-chicken-IgM-AP and immunoglobulin light chain expression by rabbit-anti-chicken-IgY-AP. Mouse-anti-β-actin followed by goat-anti-mouse-AP was used to detect β-actin. b) The cell lines from above and Igl^huVK cells with a stop codon (Igl^huVK-Stop) in CDR1 cultured for four weeks were stained with mouse-anti-chicken-IgM followed by goat-anti-mouse-Ig-Cy5. All cell lines except wild type express eGFP from the selectable marker cassette used in the knockouts. Fluorescence signal was visualized using a Beckman Coulter FC-500. c) 1×10⁶ wild type DT40 cells, non-green Igl^KO, Igh^KO cells and non-green Igl^huVK, Igh^huVH DT40 cells were labeled with FLUO-4-AM and incubated with 10 µg/ml goat-anti-chicken-IgM starting from the time point indicated by arrows. The change in fluorescence intensity was measured for a total of 300 sec using a Beckton Dickinson LSRII Fortessa. One of three representative experiments is shown. d) The same cell lines (DT40 wt grey, Igl^KO, Igh^KO red, Igl^huVK, Igh^huVH blue) were stained with goat-anti-human-kappa-RPE. Fluorescence was measured using a Beckman Coulter FC-500.