Assaying Blood Cell Populations of the Drosophila melanogaster Larva

The overall goal of this procedure is to isolate and quantify the circulating and resident blood cell populations from single drosophila larvae. This is accomplished by first gently bleeding the circulating he cytes through defined incisions in the larval body wall. The second step is to release the population of resident blood cells from the hematopoietic pockets located between the larval epidermis and muscle layers, which requires scraping or jabbing using a needle or other dissection tool.

Any hemo cytes remaining in the larval carcass are counted and the released blood cells are imaged on the glass slides where the larvae were dissected. The final step is to perform Image J analysis to quantify the released blood cells. This enables a calculation of the number of circulating and resident hemo cytes per larva, for example, comparing larvae of different developmental stages.

The fruit flight rosala melon. Augusta has been an excellent model to understand general principles in immunity and blood cell development. Blood cells and invertebrates are also called hemos, which are commonly thought to be circulating in the hemolymph.

However, at many developmental stages and in the adult, a large portion of hemo sots are actually found in Cecile clusters that are resident in certain tissues. Recent research has focused on these resident hemo sote populations, their properties and regulation, and we have developed a method that allows to selectively isolate the resident and circulating blood cells from the drosophila larva Compared to existing methods which release he cytes uns selectively. This method distinguishes between the circulating and resident populations, and it provides an automated and reproducible approach to their isolation and quantification.

We developed this method when we started to study the resident blood cells in the hematopoietic pockets of the drosophila larva. We found that when we selectively isolate resident versus circulating hemo sots, that these two populations show different behavior, such as in proliferation. Visual demonstration of this method is helpful because it is important to familiarize yourself with the location of the resident in circulating hemo cytes and how to release them selectively.

The method allows to address key questions in development, hematopoiesis and immunity, for example, regarding signals that induce blood cells and promote their localization to the hematopoietic pockets and vice versa. Regarding signals that mobilize Cecile hemos into circulation, for example, after an immune challenge, Individuals new to this method may struggle with handling the larvae gently and making precise incisions for the bleed portion of this procedure. These steps are critical to make sure that the two populations do not mix and become released at the same time.

This method is very useful for drosophila of various sizes and in stars and can be adapted to other developmental stages and potentially to other invertebrates as well. To isolate larvae from the fly food, squirt water into the vial and flush the larvae into a Petri dish. Pick larvae out of the Petri dish using a paint brush and place them into water in a cavity dish ish.

Transfer the larvae onto a slide on a cold metal block and select larvae of the desired size and genotype under a fluorescence microscope. Larvae should carry transgenes that mark he cytes by expression of a fluorescent protein or another visible he cyte marker. Place one larva into the first pap pen.

Well, to isolate circulating he cytes use dissection, scissors, or two clean needles to make incisions on the ventral side of the larva. To avoid resident, he cytes one incision at the posterior end and one incision at the anterior end of the larva. Ensure the incisions will be made in the same location on each larvae.

Allow the larvae to bleed for a few seconds without any pressure or physical agitation. After a few seconds, gently lift the larvae with the needles or forceps and place into a second. Well to rinse to release the resident he cytes.

Gently transfer the larvae to the next well and pin the larvae with one needle point. Use another needle to break up the clusters of he cytes visible through the larval body wall. Avoid the lymph gland while releasing the resident.

He cytes. Choose one part of the larvae to prod in each well and do the same for the remaining samples. Place the final carcass on a clean area of the same slide and spread it as thinly as possible to maximize the focus on the optical layers.

Count the remaining he cytes manually with a tally counter. Once the dissection is complete, weight between five to 15 minutes for the cells to settle. Before imaging, the wells store the slide in a moist chamber to avoid drying.

The settled he cytes on the slide are examined under a fluorescence microscope fluorescence images of the settled he cytes are taken for the single well method. The imaging field should cover the whole well for the tile scanning method. The margins of the wells are selected and images are acquired using commercial microscope.

Tile scan software resulting images are then analyzed for hemo site quantification. Image J software is launched and the well image is opened. Ensure that the image is eight bit or 16 bit.

Adjust the threshold by selecting image. Then select adjust and threshold. To observe the threshold window, check the dark background option and select red.

Then adjust the lower threshold level. This setting will enable the user to mark the cells with a red dot. The cells that are not being covered will be seen in gray scale.

Launch the particle analyzer to count the cells, select, analyze and click on analyze particle. Select overlay outlines to see the algorithm count to analyze the cell number, click okay and observe the summary window with the count Resident, he cytes can be mechanically disturbed, resulting in a transient increase in the circulating blood cell population to achieve disturbance. Whole larvae are vortexed with glass beads and are allowed to recover for a defined period of time during which he cytes relocate to the hematopoietic pockets.

This is illustrated by quantifying the fraction of circulating blood cells before and after disturbance and after the recovery period to mechanically disturb resident hemo cytes select up to four to eight larvae and place them in a two milliliter micro centrifuge tube with approximately 0.5 grams of 600 micron glass beads and 0.5 milliliters of water. Vortex the tube by hand at speed, 10 for one minute. Retrieve the larvae from the glass beads by spilling the contents of the micro centrifuge tube into a Petri dish and picking out the larvae with a paint brush for the recovery phase.

Place larvae in the previously prepared Petri dishes with a thin layer of fly food. Incubate the larvae for 45 minutes to allow the larvae to reestablish their hemo cyte pattern after the recovery period. Continue with the bleed scrape dissections as described previously in the control larvae, hemo cytes are localized in hematopoietic pockets, forming lateral patches and dorsal stripes.

Transient mechanical disturbance of larvae leads to a dramatic increase in the population of circulating hemo cytes at the expense of resident hemo cytes. After the recovery period, he cytes return to the hematopoietic pockets. Larvae may display enlarged dorsal vessel associated clusters and dorsal stripes, which are predominant sites of early post disturbance accumulation.

The assessed percentages of circulating hemo cytes were quantified using the bleed scrape method. This method provides a new approach for invertebrate blood cell research. It allows to investigate the population of residents self-renewing blood cells and other hemo cytes in order to understand the regulation by local microenvironments and systemic cues.

After watching this video, you should have a good understanding of how to selectively isolate and quantify circulating in resident hemos instal larva Once mastered, this technique can be completed in about 15 minutes per larva. This includes the release imaging and quantification of blood cells. Following this procedure, other methods such as immunochemistry and cell biological assays can be performed to address questions such as the status of blood cell proliferation, differentiation, survival, or phagocytosis.

While attempting this procedure, it's important to stay calm and release circulating hemo cytes with care handle larvae gently to avoid releasing resident hemo cytes along with the circulating ones.