Name: Isolating Single Cells from Xenopus Early Embryos and Sorting Them by Size (Video) | JoVE Journal
Uploaded: 2026-03-20T14:05:00.000+00:00
Duration: 4 min 33 s
Description: Learn to isolate and sort single cells from Xenopus laevis early embryos by size using strainers and density gradient separation. This method enables precise study of cell size effects in developmental biology.

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5 minutes

We would like thank the Matthew Good lab at the University of Pennsylvania for training. This work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R03HD105802).

Animal work described here has been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of South Carolina.
1. Solution preparation
NOTE: Before starting the protocol, prepare the following solutions.
<ol>
	<li>Prepare 20&#215; MMR: Mix 100 mM HEPES, 2 mM EDTA, 2 M NaCl, 40 mM KCl, 20 mM MgCl2, and 40 mM CaCl2. Adjust pH to 7.8 with NaOH. Autoclave and store at room temperature.</li>
	<li>Prepare Ca2+, Mg2+-free medium (CMFM): Mix 50.3 mM NaCl, 0.7 mM KCl, 9.2 mM Na2HPO4, 0.9 mM KH2PO4, 2.4 mM NaHCO3, and 1.0 mM EDTA. Adjust pH to 7.4, if necessary. Autoclave and store at room temperature.</li>
	<li>Prepare a 1.5% agarose gel in 60-mm Petri dish: Add 0.75 g of agarose to 50 mL of CMFM. Heat to dissolve the agarose in a microwave oven. Add 5-10 mL of agarose gel to coat a thin layer in a 60-mm Petri dish.</li>
	<li>Prepare a 15% density gradient medium (wt/vol)/CMFM: Slowly add 15 g of density gradient medium into 100 mL of CMFM. To facilitate the dissolving, heat and stir on a plate until the density gradient medium is completely dissolved. Keep the solution at 4 &#176;C until use.</li>
	<li>Prepare 4% PFA/1&#215; MEM: Mix 1 vial (10 mL) of 16% paraformaldehyde stock and 4 mL of 10&#215; MEM (1000 mM MOPS pH 7.4, 20 mM EGTA, and 10 mM MgSO4). Add ultrapure water to 40 mL. 
	NOTE: To obtain Xenopus embryos, follow a standard protocol to perform in vitro fertilization (IVF) (for example, see Sive et al., 2007)10. The embryo should be in 0.1&#215; MMR ready for use.</li>
</ol>
2. Dissociating early embryos into single cells
<ol>
	<li>Add ~15 mL of the CMFM to a new 60-mm petri dish coated with 1.5% agarose gel. Use a plastic transfer pipette to transfer 10-100 embryos (depending on the experimental needs) into the dish. Put the dish under a stereomicroscope.</li>
	<li>Use tweezers in both hands to carefully remove the vitellin membrane by catching it on the surface of the embryos with one tweezer and peeling it off with the other. Avoid damaging the embryos during the process.</li>
	<li>Incubate the devitellined embryos in the CMFM for ~1 h at room temperature. Shake the dish very gently every 5 min to facilitate the dissociation of cells. Check regularly until no piles of blastomeres are seen (Figure 1).</li>
</ol>
3. Sorting the dissociated cells by size
<ol>
	<li>During the blastomere dissociation, submerge the cell stainers (40 &#181;m, 70 &#181;m, and 100 &#181;m) in individual Petri dishes filled with CMFM (Figure 2). Place the dishes in the order from left to right as indicated. Also include two more CMFM-filled dishes between the groups for brief washing of cell stainers during each sorting.</li>
	<li>Gently swirl the dish containing the isolated single blastomeres to enrich them at the center of the dish. Carefully transfer the blastomeres into the 40 &#181;m stainer in CMFM.</li>
	<li>Gently shake the strainer a few times to let the blastomeres &#60; 40 &#181;m settle through the strainer. Then quickly move the 40 &#181;m strainer to the CMFM-filled dish for brief washing. Carefully transfer the blastomeres in the 40 &#181;m strainer, which are &#62; 40 &#181;m, into the 70 &#181;m strainer.</li>
	<li>Repeat the steps until the blastomeres are sorted in the 100 &#181;m strainer.</li>
	<li>Gently swirl each dish with sorted blastomeres to enrich them at the center of the dish. Carefully transfer the blastomeres (~4 mL) onto the top of 0.5 mL of 15% density gradient medium in a fluorescence-activated cell sorting (FACS) tube (Figure 3). Avoid mixing.</li>
	<li>Let the tube sit for 10 min. The intact blastomeres will settle down to the bottom by gravity, separating from the yolk globules from the burst blastomeres during the sorting and transfer (Figure 3).</li>
	<li>Depending on the purpose of downstream experiments, proceed with live cell analysis, freeze the blastomeres in liquid nitrogen for RNA work, or fix the blastomeres for immunostaining (Figure 3).</li>
</ol>

<ol><li>Ginzberg, M. B., Kafri, R., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+biology.+On+being+the+right+%28cell%29+size%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Cell biology. On being the right (cell) size.</a> Science. 348 (6236), 1245075(2015).</li>
<li>Xie, S., Swaffer, M., Skotheim, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eukaryotic+cell+size+control+and+its+relation+to+biosynthesis+and+senescence%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">Eukaryotic cell size control and its relation to biosynthesis and senescence.</a> Annu Rev Cell Dev Biol. 38, 291-319 (2022).</li>
<li>Chadha, Y., Khurana, A., Schmoller, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eukaryotic+cell+size+regulation+and+its+implications+for+cellular+function+and+dysfunction%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Eukaryotic cell size regulation and its implications for cellular function and dysfunction.</a> Physiol Rev. 104 (4), 1679-1717 (2024).</li>
<li>Chen, H., Einstein, L. C., Little, S. C., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+patterning+of+zygotic+genome+activation+in+a+model+vertebrate+embryo%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo.</a> Dev Cell. 49 (6), 852-866.e7 (2019).</li>
<li>Hubatsch, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cell+size+threshold+limits+cell+polarity+and+asymmetric+division+potential%5BTitle%5D)+AND+%22Nat+Phys%22%5BJournal%5D)">A cell size threshold limits cell polarity and asymmetric division potential.</a> Nat Phys. 15 (10), 1075-1085 (2019).</li>
<li>Fung, H. F., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Function+follows+form%3A+How+cell+size+is+harnessed+for+developmental+decisions%5BTitle%5D)+AND+%22Eur+J+Cell+Biol%22%5BJournal%5D)">Function follows form: How cell size is harnessed for developmental decisions.</a> Eur J Cell Biol. 102 (2), 151312(2023).</li>
<li>Chen, H., Qian, W., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+cellular+dimensions+with+cell+differentiation+during+early+development%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">Integrating cellular dimensions with cell differentiation during early development.</a> Curr Opin Cell Biol. 67, 109-117 (2020).</li>
<li>Clute, P., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+appearance+of+division+asynchrony+and+microtubule-dependent+chromosome+cycles+in+Xenopus+laevis+embryos%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Regulation of the appearance of division asynchrony and microtubule-dependent chromosome cycles in Xenopus laevis embryos.</a> Dev Biol. 171 (2), 273-285 (1995).</li>
<li>Venkataraman, T., Dancausse, E., King, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PCR-based+cloning+and+differential+screening+of+RNAs+from+Xenopus+primordial+germ+cells%3A+Cloning+uniquely+expressed+RNAs+from+rare+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">PCR-based cloning and differential screening of RNAs from Xenopus primordial germ cells: Cloning uniquely expressed RNAs from rare cells.</a> Methods Mol Biol. 254, 67-78 (2004).</li>
<li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenopus+laevis+in+vitro+fertilization+and+natural+mating+methods%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">Xenopus laevis in vitro fertilization and natural mating methods.</a> CSH Protoc. 2007, (2007).</li>
</ol>

The author declares no competing interests.

Cell size is fundamentally important for cellular functions3, and its impact on early development requires more scrutiny in model embryonic systems. Here we have described the details for isolating single cells from Xenopus laevis early embryos and sorting the cells by size using cell strainers, potentially applicable for cell size studies. This method has allowed us to provide the direct evidence of size-regulated zygotic gene activation in early embryogenesis4.</...

Cell size change is a common feature of early embryogenesis, when the fertilized egg undergoes multiple rounds of cleavage, generating an increasing number of cells. While much is known about the role of cell size as a major parameter controlling cellular functions1,2,3, how cell size impacts early development is less known. Recent work across various model embryonic systems suggests that cell size regulates zygotic genome activation (ZGA)4 and cell fate specification5, supporting an important role for cell size in developmental decisions6,7.
The Xenopus early embryo is an important model for studying cell size-regulated developmental decision-making. Because the Xenopus early embryo is large (~1.2 mm in diameter), it is easy to manipulate its size physically, making it useful for assessing the direct consequences of cell-size change. For example, mini embryos of varying sizes can be generated by constriction of 1-cell embryos using hair loops4,8, demonstrating dose-dependent ZGA4. Moreover, Xenopus early embryo contains a gradient of cell sizes along the animal-vegetal axis, with the smallest cells at the animal pole and the largest cells at the vegetal pole, demonstrating an over 100-fold difference in volume4. Importantly, by directly visualizing and quantifying nascent transcripts in single cells of whole-mount embryos, we identified a cell-size threshold that regulates ZGA4. Further, isolation of single cells from Xenopus early embryos and sorting them by size confirmed the importance of the cell size-threshold ZGA4.
Here we describe the detailed procedures for isolating single cells from Xenopus laevis early embryos and sorting them by size. Xenopus early embryos can be dissociated into individual cells by incubating them in Ca2+, Mg2+-free medium (CMFM)9, and these single cells can further be sorted using cell strainers with varying mesh pore sizes. The sorted cells can be purified and used in various applications, including live cell analysis, sequencing, or imaging assays.

<table><tbody><tr><td>16% Paraformaldehyde (PFA)</td><td>EMS</td><td>15710-S</td><td>For making the fixative solution</td></tr><tr><td>60 mm Disposable Petri Dishes</td><td>VWR</td><td>25384-092</td><td>For incubating embryos</td></tr><tr><td>ACCU-SCOPE 3075 Zoom Stereomicroscopes</td><td>VWR</td><td>470351-130</td><td>For visualizing embryos</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A-9414</td><td>For coating petri dishes</td></tr><tr><td>Cell Strainer, 100 &amp;micro;m</td><td>VWR</td><td>76327-102</td><td>For sorting cells by size</td></tr><tr><td>Cell Strainer, 40 &amp;micro;m</td><td>VWR</td><td>76327-098</td><td>For sorting cells by size</td></tr><tr><td>Cell Strainer, 70 &amp;micro;m</td><td>VWR</td><td>76327-100</td><td>For sorting cells by size</td></tr><tr><td>Disposable Transfer Pipettes</td><td>VWR</td><td>414004-035</td><td>For transferring embryos</td></tr><tr><td>Dumont Tweezer, Style 5</td><td>Electron Microscopy Sciences</td><td>72701-D</td><td>For removing vitelline membrane</td></tr><tr><td>Glass Petri Dish, 60 &amp;times; 15 mm</td><td>VWR</td><td>75845-542</td><td>For incubating embryos</td></tr></tbody></table>

isolating single cells from xenopus early embryos and sorting them by size

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

Early embryogenesis requires an exponential decrease in cell size, while the embryo remains constant in size at the earliest stages of development. The functional importance and the mechanisms by which cell size impacts embryo development are still largely understudied. The Xenopus early embryo&#160;provides a unique system to address these major gaps. Compared with other model organisms, the Xenopus early embryo is extremely large and contains a cell-size gradient, from small cells at the animal pole to large cells at the vegetal pole, across the entire embryo at stages around the mid-blastula transition. Here, we describe a protocol for dissociating single cells from Xenopus early embryos and sorting them by size using cell strainers with varying mesh pore sizes. As determined by imaging and size measurements, the sorted cells are within the expected size range and can be used for live-cell analysis or sequencing, or fixed for imaging. This system will shed new light on revealing size-dependent regulatory mechanisms during early development.

Xenopus laevis embryos (n = 50) at Stage 9 were incubated in the CMFM medium to dissociate into single blastomeres following this protocol (see an example in Figure 1). The dissociated single blastomeres were sorted sequentially using the 40 µm, 70 µm, and 100 µm cell strainers as described above. The unsorted control mix and he sorted single blastomeres were fixed in 4% paraformaldehyde and imaged under a bright-field microscope (Figure 4A). As shown i...

We describe a method for isolating single cells from Xenopus laevis early embryos and sorting them by cell size.

Early embryogenesis requires an exponential decrease in cell size, while the embryo remains constant in size at the earliest stages of development. The ...

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.

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Learn to isolate and sort single cells from Xenopus laevis early embryos by size using strainers and density gradient separation. This method enables precise study of cell size effects in developmental biology.

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

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