Name: using flow cytometry to detect and quantitate altered blood formation in the developing zebrafish
Uploaded: 2021-04-29T14:00:00
Description: Watch this Scientific Journal Video about Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish at JoVE.com

Funding was provided by the National Institutes of Health (NIH: R15 DK114732-01 to D.L.S.), the National Science Foundation (NSF: MRI 1626406 to D.L.S.), and from the Honor&#39;s Program at California State University Chico (to K.F.R.). The authors thank Betsey Tamietti for laboratory management and Kathy Johns for administrative assistance.

The Institutional Animal Care and Use Committee (IACUC) advisory board at California State University, Chico, approved all methods described below.
NOTE: It is advised to treat embryos with 1-phenyl 2-thiourea (PTU; Table 1) at 24 hours postfertilization (hpf) to prevent pigmentation which negatively affects fluorescence discrimination by flow cytometry. All procedures listed below are acceptable for flow cytometry and fluorescence-activated cell sorting (FACS). However, if on...

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Zebrafish are an excellent model system for studying primitive and definitive vertebrate hematopoiesis. Over the past few decades, multiple assays have been developed and refined, allowing zebrafish to become a quick and economical model for testing drugs, generating and testing genetic mutants, and overall allowing researchers to analyze molecular pathways essential for hematopoiesis. This protocol utilizes embryonic zebrafish which allows quick data collection, the use of less physical space than adult animals, and the...

Blood production (hematopoiesis) is an essential developmental process that first starts in the early embryo. This process begins by generating primitive red blood cells and macrophages directly from mesoderm, and later shifts towards the production of hematopoietic stem and progenitor cells (HSPCs). These stem cells, which are multipotent, generate all of the varieties of mature blood cells in the organism. Capable of self-renewal, the system is continually replenished through these HSPCs. While this process starts early in development, hematopoiesis continues for the life of the animal, providing the ability to transport oxygen to distant sites of the body, to stop bleeding after injury, and to protect the body from infection. The development of this complex system is controlled temporally and spatially during development and any perturbations in blood cell production can be catastrophic for the organism, resulting in anemia, thrombocytopenia, leukopenia, and leukemia.
A popular animal model used for hematopoietic research is the zebrafish (Danio rerio) because they have similar blood development when compared to humans. In fact, many of the genes and molecular pathways used during hematopoiesis are conserved throughout vertebrate evolution, allowing us to learn about human genes by studying zebrafish. Importantly, zebrafish embryos develop outside the body and within 7 days have generated most mature blood cell types, allowing for direct visualization of the hematopoietic system in a short amount of time. Zebrafish are also extremely fecund, which allows researchers to observe a larger number of samples in a short time frame, which is also important for generating reproducible data. The zebrafish&#39;s short generation time and external development provides for easier manipulation and observation during mutagenesis studies1,2,3,4,5 and drug screening6,7,8,9,10. This allows a panel of promising therapeutic compounds for human blood disorders to be quickly and efficiently tested.
Importantly, zebrafish are also genetically amenable, and the genome is sequenced and annotated. This tractability allows reverse genetics techniques such as morpholino- (MO-) mediated knockdown and CRISPR-mediated genetic ablation to be performed. Zebrafish have also proven their utility as a model to perform forward genetic screens; many essential genes and pathways involved in vertebrate blood formation have been discovered in this manner. Numerous methods of observing blood cells have also been developed in zebrafish. While traditional histological staining techniques exist, it is also possible to perform in situ hybridization for blood-specific transcripts. Importantly, numerous transgenic lines of fish also exist whereby fluorescent proteins are expressed by lineage-specific promoters, allowing the labelling of specific blood cells with fluorescent proteins11. This allows researchers to perform up-to-the-minute observation of blood cell genesis, expansion, and regulation in a living organism over time.
Overall, conservation of the hematopoietic system, the presence and easy development of transgenic lines, easy visualization, and short generation time has made the zebrafish an economical, fast, and adaptable model of hematopoiesis. To improve upon the toolbox of techniques available for zebrafish researchers, we developed this assay to robustly quantitate the number of blood cells in embryos. The method involves digesting transgenic animals and performing flow cytometry for fluorescent blood cells. In this way, blood cells from mutant animals, the effect of MO and CRISPR modification, and the effect of small molecules can be quantitatively analyzed in a quick and reproducible manner. These assays are user-friendly and economical ways to enumerate blood cells, allowing examination of their generation, proliferation, and maintenance over time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 ml MCF tube</td>
			<td>FisherBrand</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm Polystyrene easygrip Petri dish</td>
			<td>Corning Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene round bottom tube with cell strainer cap</td>
			<td>Corning Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria Fusion flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiolthreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (10x) with Ca2+ and Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS 500 mL</td>
			<td>Gemini Bio-Products</td>
			<td>100-108</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone PBS (1x)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>sh30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Librease</td>
			<td>Roche Sigma-Aldrich</td>
			<td>5401119001</td>
			<td>dissociation protease</td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Roche Sigma-Aldrich</td>
			<td>11459643001</td>
			<td>dechorionation protease</td>
		</tr>
		<tr>
			<td>SYTOX Red Dead Cell Stain</td>
			<td>Invitrogen&#160;</td>
			<td>S34859</td>
			<td></td>
		</tr>
	</tbody>
</table>

using flow cytometry to detect and quantitate altered blood formation in the developing zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

To enumerate red blood cells in embryonic zebrafish, lcr:GFP16 embryos were injected at the one-cell-stage with PBS, 7.0 ng/nL ism1 MO, or 7.0 ng/nL ism1 MO with 7.0 ng ism1 mRNA12. At 48 hpf they were digested and subjected to flow cytometry analysis. After analyzing the percentage of GFP+ red blood cells from each sample (each sample is 5 randomly selected embryos; Figure ...

Watch this Scientific Journal Video about Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish at JoVE.com

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

This protocol is significant because it allows the knockdown and over expression of genes in a developing fish, and the quantitation of those downstream effects on blood cells. This protocol is quick, easy to perform, economical, and easy to automate with access to the proper instrumentation. Demonstrating this procedure will be Kristen Rueb, an undergraduate researcher in the laboratory.Begin by transferring between five and 200, 48 hours post fertilization embryos into a plastic 10 centimeter Petri dish. Tilt the dish so the embryo sink to the bottom edge, and remove as much E3 medium from the dish as possible. Then add 500 microliters of dechorionation protease to the embryos.After five minutes at room temperature, tip all of the embryos to the bottom edge of the plate again and gently tap the side of the dish, allowing the embryos to gently rub against the bottom of the plate to completely remove their chorions. Using a squeeze bottle add approximately 20 milliliters of E3 medium to dilute the protease, and allow the embryos to settle. Then decant the medium, and rinse the embryos three more times with fresh medium as just demonstrated to remove all traces of the protease.To prepare the embryos for dissociation, use a P1000 pipette to transfer five to 10 embryos into one, 1.5 milliliter micro centrifuge tube, and aspirate the transferred E3 medium. Then add one milliliter of 10 millimolar DTT in E3 medium to the embryonic zebrafish, and place the microcentrifuge tube in a horizontal position for 30 minutes at room temperature to remove the mucus coating. For embryo dissociation, wash the DTT treated embryos three times with one milliliter of DPBS supplemented with calcium and magnesium per wash.Before adding 500 microliters of DPBS supplemented with calcium and magnesium, and five microliters of five milligrams per milliliter, dissociation protease. Then incubate the samples at 37 degrees Celsius on a horizontal orbital shaker at 185 revolutions per minute for 60 minutes. Be sure to periodically check on the embryos, so you don't over digest them.When a not completely homogeneous sample with some solid tissue present can be observed, triturate the embryos with a P1000 pipette until the sample is fully dissociated. To prepare the dissociated zebrafish embryonic cells for flow cytometry, transfer the entire cell sample onto the top reservoir of a five milliliter polystyrene round bottom tube, with a 35 micrometres cell strainer cap. Rinse the cap with four milliliters of PBS without calcium or magnesium, and pellet the cells by centrifugation.Then resuspend the cells in 500 microliters of PBS without calcium and magnesium per tube. For flow cytometric analysis of the zebrafish embryonic cells, gently vortex the cell suspensions before adding a one-to-one thousand dilution of red dead cell stain to each tube. Within 30 minutes of applying the dead cell stain, empty the waste tank, fill the sheath tank with the appropriate solution, and start the fluidic system of the flow cytometer.In the analysis software, draw five plots, and set the first plot to measure forward versus side scatter. Set the forward scatter to linear, and the side scatter to logarithmic. Set the second plot to measure forward scatter height versus forward scatter width, and the third plot to measure scatter height versus side scatter width.Set the fourth plot to measure the red dead cell stain on the x-axis, and side scatter on the y-axis to allow the discrimination of live from dead cells. The fifth plot should be set to measure the fluorophore of choice. When all of the plots have been set, load the sample onto the cytometer and reduce the flow rate so that the cells do not run out to quickly.Adjust the forward and side scatter settings so that the bulk of the cell population can be clearly observed, and draw a gate around the cell population. Label this gate cells. With the second dot plot gated on the cells, gate on forward scatter and the side scatter singlets to exclude doublets.In the fourth plot, adjust the red dead cell stain settings so that there is a clearly negative population. Draw a gate around these cells and label this gate live cells. In the fifth plot, gate on the live cells and draw a gate around the positive cells, then run each sample collecting at least 25, 000 live cell events.When all of the samples have been analyzed, follow the shutdown procedure to turn off the fluidics, refill the sheath tank and empty the waste tank. After analyzing the percentage of GFP positive red blood cells from each embryonic zebrafish sample, the average of all the control group can be calculated. Typically, the average is set as one and all of the percentages are calculated from this reference point.In this representative analysis, it was determined that reducing the ISM1 transcript with a specific morpholino, reduce the number of GFP positive red blood cells present within 48 hours post fertilization embryos. Rescuing this reduction in ISM1 morpholino levels with exogenous mRNA, return the number of red blood cells to normal. Proper digestion on the samples is critical.If you over-digest or under-digest, you will not get the correct results. If a fax machine is available, researchers can collect the blood cells by sorting for in vitro culture, RNA sequencing and quantitative RT-PCR.

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