Fig 1.
Detection of MMc cells from a whole embryo.
(A) Female mice heterozygous for the GFP locus (GFP+/-) were mated with non-GFP-carrying wild-type male mice, and offspring with no GFP gene (GFP-/-) were analyzed for GFP+/- MMc cells. (B) Bright-field and fluorescent-field images showed which fetus inherited the GFP gene (right) and which did not (E18.5, litter). GFP-/- fetus was used for MMc cell isolation. (C) Schematic illustration of the developed method for detecting MMc cells from a whole embryo (see also Method). To avoid cross-contamination of maternal cells, an isolated embryo was washed three times in cold phosphate-buffered saline. The embryo was then minced and incubated in an enzyme cocktail, followed by two (rough and fine) filtrations to dissociate cells. Rough filtration was carried out to dissociate the solid tissues, and fine filtering was carried out to generate a single cell suspension. From this single cell suspension, live MMc cells were counted and isolated using FACS.
Fig 2.
Dissociation of cells by filtration and survival rate of isolated cells.
(A) Minced samples before and after the rough filtration are shown for each developmental stage. A 100-μm cell strainer was used for the rough filtration. Note that more aggregates remained in samples from the later stages E16.5 and E18.5. (B) Samples after fine filtration with the 70-μm cell strainer. (C) Rough filtered samples were stained with trypan blue, and finer filtration was performed to estimate whether the aggregates were mainly composed of dead cells. (D) Live cell rate in each developmental stage. Calculation of live cell rate (%) in the cell suspensions was evaluated using PI marker (dead cell marker) and FACS. The control sample originated from an adult spleen. Each circle represents the ratio of live cells in a single sample. [control: n = 11, E12.5: n = 1 (68.8%), E13.5: n = 2 (median 84.3%), E14.5: n = 5 (median 78.4%), E15.5: n = 4 (median 97.0%), E16.5: n = 2 (median 95.4%), E18.5: n = 4 (median 87.0%)].
Fig 3.
FACS detection of MMc cells from individual embryos.
(A) Gating conditions for counting MMc cells. After the rough and finer filtration of the minced embryo, isolated cells from a single GFP-/- embryo were analyzed using FACS. Mother blood cells were used as positive control, and cells from a wild-type fetus with a wild-type mother were used as negative control. Noise and/or debris were removed with gate P1, and then doublet cells were removed with gate P2 and P3. Gate P4 (GFP+ PI-) was defined using positive control cells and applied to sort the GFP+, potential MMc cells. (B) For each sample (individual embryo), the numbers of sorted MMc cells were counted as GFP+ cells, and the ratios over the 107 sorted cells were calculated. Some GFP+ cells were detected for the negative control samples (wild-type embryo with wild-type mother). Sample numbers are as follows. control: E12.5: n = 5, E13.5: n = 5, E14.5: n = 5, E15.5: n = 5, E16.5: n = 5, E18.5: n = 4, sample: E12.5: n = 6, E13.5: n = 7, E14.5: n = 4, E15.5: n = 5, E16.5: n = 7, E18.5: n = 4. See also Table 1 for more detail. (C) GFP signals of sorted cells were further analyzed using fluorescent confocal microscopy (Zeiss LSM710). Together with the sample cells, negative (wild-type cells sorted with gate P5) and positive control (mother blood cells sorted with gate P4) cells were also analyzed using microscopy.
Table 1.
Detailed information of GFP+ and GFP- fetal samples in Fig 3B.