Colony forming abilities are reduced when CreERT2 positive cells from young mice are cultured with 4-OH tamoxifen. Young female CreERT2 positive marrow cells form more colonies than CreERT2 negative cells in the absense of 4-OH tamoxifen, and young female marrow CreERT2 negative cells cultured with 4-OH tamoxifen give rise to more colonies than when cultured with the ethanol vehicle control.
(A) Brightfield images of representative colonies from female marrow cells cultured under hypoxia (scale bar = 5mm). (B) The colony forming assays were performed by culturing cells from central marrow and enzymatically digested flushed long bone for 10 days following culture in media with 4-OH tamoxifen (1μM) or vehicle control ethanol. Mean (±SEM) CFU-F assay colony numbers are reduced following CreERT2 activation with 4-OH tamoxifen in all Cre+ marrow and bone cells, irrespective of sex or culture conditions, compared to culture with ethanol. Mean (±SEM) CFU-F assay colony numbers are also reduced following CreERT2 activation with 4-OH tamoxifen in Cre+ male marrow and bone cells, and female bone cells, compared to Cre- culture with 4-OH tamoxifen. Mean (±SEM) CFU-F assay colony numbers are higher from female Cre+ marrow cells cultured under both normoxia and hypoxia with ethanol, compared to Cre- marrow cells with ethanol. Mean (±SEM) CFU-F assay colony numbers are increased in female Cre- marrow cells cultured with 4-OH tamoxifen compared to ethanol, when cultured under both normoxia and hypoxia. *p<0.05 **p<0.01 ***p<0.001. (n = 3 experiments. All experiments were performed in technical triplicate). (y axis = mean number of colonies with diameter greater than 1mm).