Eosinophils can be cultured from frozen bone marrow cells and in a 96-well plate.

<p>(A) Histogram (left panel) of cultured eosinophils that had been frozen, thawed and then stained with viability dye is shown. Percentages of viable (negative for the dye) and dead (positive for the dye) eosinophils recovered from the frozen vial are indicated in the histogram. Surface expression of CCR3 and Siglec-F (right panel) on all cells recovered from the frozen vial is shown in a representative density plot from 4 independent experiments. (B) Total number of (mean ± SD, <i>n</i> = 3 wells per condition) Day 15 eosinophils (Fresh eosinophils) or Day 14 eosinophils that had been frozen and then thawed the next day (Frozen eosinophils) that migrated toward CCL11 at the indicated doses (a representative of 2 independent experiments is shown). ***<i>P</i><0.001, ^#<i>P</i><0.0001 vs. fresh eosinophils of the same treatment group. (C) Eosinophil yield (mean ± SEM, <i>n</i> = 3 independent experiments with 3 wells per condition per experiment) at Day 14 from cultures started with freshly harvested low-density bone marrow (Fresh LDBM, black bar) or frozen/thawed LDBM (Frozen LDBM, white bar). (D) Eosinophil yield (mean ± SD) at Day 14 from cultures started with LDBM that was freshly prepared (Fresh LDBM), was prepared from whole bone marrow (WBM) that had been frozen and thawed (Frozen WBM), was prepared from WBM and then frozen and thawed (Frozen LDBM), and was prepared from freeze-thawed WBM and then frozen and thawed again (Frozen WBM/LDBM) is shown. Data are a representative of 2 independent experiments with 3 wells per condition per experiment. (E) Total number of eosinophils (mean ± SD, <i>n</i> = 3 wells per condition) that migrated toward CCL11 at the indicated doses (a representative of 2 independent experiments is shown). **<i>P</i><0.01, ***<i>P</i><0.001, ^#<i>P</i><0.0001 when compared to eosinophils from Fresh LDBM of the same treatment group. (F) Eosinophil yield (mean ± SEM) at Day 14 in flat- or round-bottomed, 96-well plates from cultures started with LDBM cells at the indicated concentrations (<i>n</i> = 3 independent experiments with 3 wells per condition per experiment). *<i>P</i><0.05, ***<i>P</i><0.001. CCL11, C-C motif chemokine 11; CCR3, C-C chemokine receptor type 3; Siglec-F, sialic acid–binding immunoglobulin-like lectin F.