Isolating Single Cells from Xenopus Early Embryos and Sorting Them by Size

Our research focuses on how cell size regulates genome and sulfate decisions in early embryo development. Existing methods overlook size heterogeneity in early xenopus embryos. This protocol enables precise physical separation and sorting by cell size.

To begin, add approximately 15 milliliters of CMFM to a new 60 millimeter Petri dish coated with 1.5%AGROS gel. Using a plastic transfer pipette, transfer 10 to 100 Xenopus laevis embryos into the dish. Place the dish under a stereo microscope.

Carefully catch the vitelline membrane on the surface of the embryos with one tweezer and peel off the membrane with another tweezer. Avoid damaging the embryos during this process. Incubate the devitallined embryos in CMFM for one hour at room temperature.

Gently shake the dish every five minutes to facilitate cell dissociation. Check regularly until no piles of blastomeres are visible. During blastomere dissociation, submerge the 40 micrometer, 70 micrometer, and 100 micrometer cell strainers in individual Petri dishes filled with CMFM.

Arrange the dishes from left to right in the indicated order. Place two additional CMFM filled dishes between the groups for brief washing of the strainers during sorting. Now gently swirl the dish containing the isolated single blastomeres to enrich them at the center.

Then carefully transfer the blastomeres into the 40 micrometer strainer submerged in CMFM. Gently shake the strainer a few times to allow blastomeres smaller than 40 micrometers to pass through. Quickly move the strainer to a CMFM filled dish for brief washing.

Then carefully transfer the blastomeres retained in the strainer into the 70 micrometer strainer. Repeat the sorting procedure using progressively larger strainers until the blastomeres are collected in the 100 micrometer strainer. Gently swirl each dish containing sorted blastomeres to enrich them at the center.

Then carefully transfer approximately four milliliters of blastomeres onto the top of 0.5 milliliters of 15%density gradient medium in a fluorescence activated cell sorting tube without mixing. After 10 minutes, the intact blastomeres will settle to the bottom by gravity, separating them from yolk globules derived from burst blastomeres. The unsorted control and size sorted single blastomeres were fixed and imaged under a bright field microscope.

The sorted blastomeres showed narrow size distributions within their respective expected ranges relative to the control unsorted blastomeres. The less than 40 micrometers group showed a mean diameter of approximately 33 micrometers. The 40 to 70 micrometers group showed a mean diameter of approximately 55 micrometers, while the larger than 70 micrometers group showed a mean diameter of approximately 91 micrometers.

This protocol allows researchers to study functions and mechanisms mediated by cell size. The most important consideration for this protocol is to carefully handle the dissociated cells as they are fragile. After this protocol, the sorted cells can be used for cell cycle analysis, single cell sequencing, and confocal imaging.