Noninvasive Intravascular Microtransfusion in Colonial Tunicates

Abstract

Tunicates are a diverse group of worldwide marine filter-feeders that are vertebrates’ closest invertebrate relatives. Colonial tunicates are the only know chordates that have been shown to undergo whole-body regeneration (WBR). Botrylloides in particular can regenerate one fully functional adult from a minute fragment of their vascular system in as little as 10 days. This regenerative process relies on the proliferation of circulating stem cells, likely supported by the activity of some of the 11 identified types of hemocytes. To study and challenge WBR, it is thus important to have the capacity to isolate, analyze, and manipulate hemolymph in regenerating colonies. Here we present a microtransfusion technique that permits the collection of pure hemocytes, the quantification of their purity, their labeling, and reinjection into colonial tunicates. To exemplify our approach, we present in addition a protocol to analyze the isolated hemocytes using flow cytometry. Our approach is minimally invasive, does not induce lethality, and therefore allows repeated transfusion into exactly the same colony with minimal disruption to the process being studied.

1 Introduction

Tunicates are a group of worldwide highly diverse marine invertebrates separated into three classes that include over 3000 known extant species [1, 2]. Most tunicates are benthic sessile animals that reproduce sexually through a motile tadpole larval stage [3, 4]. In addition, a number of tunicate species are able to reproduce asexually through budding [5, 6]. Budding in tunicates typically leads to the formation of colonies where animals, called zooids, are interconnected by an external vascular system (Fig. 1a). Furthermore, some species of colonial tunicates are able to undergo whole-body regeneration (WBR) [7,8,9]. This is the only known occurrence of WBR among chordates [10].

Fig. 1

Collecting hemocytes in the vascular system of Botrylloides diegensis . (a) A top-view of a colony of Botrylloides diegensis (left) and Botryllus schlosseri (right) growing on a microscopy glass slide. (b) Microinjection setup with a colony holder, a stereoscope, a microinjector, and the micromanipulator. (c) Close-up of the microtransfusion setup, with a colony placed on the colony holder and the marked micropipette close to the insertion point. (d) The external vasculature of the colony. (e) Magnification of the area delimited by the white rectangle in (d). Good candidate points for transfusion are indicated using black arrows, subendostylar points using gray arrows, and bad transfusion points using white arrows. (f) Microcollection of hemocytes. Micropipette is inserted inside the vessel lumen through the tunic and hemocytes (arrows) are flowing in. (g) Average collection rate over time (n = 5). Changes in collection point are indicated using circles, while changes in micropipette using triangles. Display are the individual collections (thin), the average collection (thick), and the corresponding standard deviation (gray)

In the Botrylloides genus, WBR is completed in as little as 10 days following the isolation of a fragment of their external vascular system [11]. WBR starts with the healing of the injury sites followed by a major remodeling of the isolated vascular system [12]. Circulating stem cells are then mobilized to regeneration niches, i.e., a discrete regeneration locus within the vascular system. These niches compete through a yet undetermined process that consistently leads to the development of a single adult zooid, while all other niches are resorbed by the vascular system [12].

In addition to this role during regeneration [12], hemocytes are involved in numerous biological processes in colonial tunicates, including immune response [13,14,15], allorecognition [16,17,18], and asexual reproduction [19,20,21]. Colonial tunicates’ hemolymph is typically composed of less than a dozen 4- to 25-μm-wide cell types classified into four functional classes: undifferentiated, phagocytic, cytotoxic, and storage cells [22,23,24,25]. For instance, in Botrylloides leachii , 11 different cell types ranging from 5 to 20 μm have been described [12], as well as the two additional mast-like and transport functional classes. Hemolymph extraction, manipulation, and alteration are thus essential tools for analyzing as well as challenging the functions of hemocytes. A large palette of approaches have been established for this purpose in colonial tunicates. Extracted hemolymph has been used for starting primary cultures of hemocytes [26], its cell composition studied through histological staining [12] as well as flow cytometry [27], and specific hemocyte populations sorted by fluorescently activated cell sorting [28]. Hemocytes have been labeled and injected in a recipient colony [29, 30], a variety of staining solutions delivered into the vasculature [31,32,33] and functional characterization undertaken by injection of small interfering RNA probes [33, 34] as well as chemical compounds [35, 36].

The most common approach for performing hemolymph collection is by mechanical injury of the vessel of an anti-clotting-treated colony. Hemolymph is then collected with a syringe or a glass micropipette as it bleeds out from the colony [12, 14, 22]. When larger amounts of hemolymph are required, mechanical dissociation of the entire colony is often used [21, 28,29,30, 37]. In both the approaches, the colony thus bears severe injuries and material exogenous to the hemolymph can contaminate the samples.

Here we describe a microtransfusion technique that allows to collect hemolymph intravascularly with high purity. We also present methods to characterize cytologically the hemolymph, label its hemocytes, and reinject them using the same setup used for the collection . This process can be routinely performed in the same colony with minimal damage, making it useful for long-term in vivo experiments aimed to study the role of the hemolymph in colonial tunicates.

2 Materials

All reagents are prepared with deionized water and stored at room temperature unless otherwise stated.

• 1. 10× K-depleted phosphate-buffered saline (K-PBS): 90 g NaCl, 10.6 g NaH₂PO₄•H₂O, 3.3 g Na₂HPO₄ in 1 L H₂O (see Note 1). Adjust pH to 7.8 with NaOH. Use within 2 months.

• 2. 3.3× K-PBS: 300 mL 10× K-PBS, 600 mL H₂O (see Note 2).

• 3. Marine Anti-Clotting solution (MAC): 0.38% (w/v) sodium citrate-NaOH, 10 mM l-cysteine, pH 7.5, in 3.3× K-PBS. Prepare fresh.

• 4. MAC-BSA-EDTA solution (MBE): 0.1% (w/v) bovine serum albumin (BSA), 10 mM ethylenediaminetetraacetic acid (EDTA) in MAC, 0.22-μm sterile filtered. Prepare fresh (see Note 3).

• 5. Purity assessment solution (PAS): 1% (v/v) Brilliant Blue FCF in MAC (see Notes 4 and 5).

• 6. Microtransfusion setup: Stereoscope, micromanipulator, microinjector capable of uptake (e.g., Cell-Tram Air, Eppendorf).

• 7. Collection support: Glass petri dish, high-profile 3D-printed colony holder (Fig. 1b, see Note 6).

• 8. Glass capillaries: Borosilicate glass capillaries compatible with the microinjector, 0.76 mm internal diameter (see Note 7).

• 9. Glass micropipettes: glass capillaries pulled using a microforge (e.g., Micropipette Puller P-87, Sutter Instrument), marked each 5 μL (i.e., each 11.02 mm for 0.76 mm ID capillaries).

• 10. Hematocytometer.

• 11. 2× brightfield cell viability dye (e.g., 0.4% (w/v) Trypan blue in 3.3× K-PBS).

• 12. Purity scale (see Note 8): 100% (100 μL MBE), 90% (90 μL MBE, 10 μL PAS), 70% (70 μL MBE, 30 μL PAS), 50% (50 μL MBE, 50 μL PAS), 30% (30 μL MBE, 70 μL PAS), 0% (100 μL PAS).

• 13. Microvolume spectrophotometer (e.g., NanoDrop, Thermo Fisher Scientific).

• 14. 200× live labeling dye (e.g., MemGlow 640, Cytoskeleton) (see Note 9).

• 15. 20× cell viability dye (e.g., 10 μg/mL Calcein Violet, eBioscience).

• 16. 500× DNA live labeling dye (e.g., 5 mM DRAQ5, Thermo Fisher Scientific).

• 17. 40-μm cell strainer (e.g., pluriStrainer, pluriSelect).

• 18. Calibration fluorescent beads (e.g., QbSure, Cytek).

• 19. 2× Fixation solution: 2% (w/v) C₁₂H₂₂O₁₁ (sucrose), 2% (v/v) C₅H₈O₂ (glutaraldehyde) in 3.3× K-PBS.

3 Methods

3.1 Intravascular Collection of Hemolymph

This protocol has been developed for Botrylloides leachii colonies, but has been successfully applied to Botryllus schlosseri as well. We thus expect it to work similarly well in all colonial ascidian species presenting an external vascular system accessible through a moderately soft tunic (see Note 10). This protocol typically yields in 30 min 40 μL (Fig. 1f) of 93% pure hemolymph (Fig. 2c) containing >99% viable hemocytes at a concentration around 300,000 cells/mL (see Note 11).

• 1. Select a colony to be used for hemolymph collection (see Note 12).

• 2. Transfer the colony on its transparent support into a container filled with 3.3× K-PBS.

• 3. Brush the colony with a fine paint brush under a stereoscope to remove debris and dirt.

• 4. Transfer the colony to another clean container filled with 3.3× K-PBS.

• 5. Trim a glass micropipette using a sharp razor blade to obtain a tip diameter of 17–30 μm (see Note 13).

• 6. Load 200 μL of MBE into a cut P200 pipette tip (see Note 14).

• 7. Insert the rear opening of the trimmed glass micropipette into the pipette tip.

• 8. Load the glass micropipette by slowly pipetting the MBE into it.

• 9. Remove the glass micropipette from the tip.

• 10. Mount the loaded glass micropipette into the holder of the microinjection device following the manufacturer’s instructions.

• 11. Assemble the needle onto the micromanipulator.

• 12. Pipette 200 μL of MBE in the 1.5-mL tube to be used for collecting the hemocytes.

• 13. Vortex the tube for 5 s to coat its internal surface.

• 14. Wait 5 min for the coating to take effect.

• 15. Remove the MBE from the tube.

• 16. Transfer the colony into a container filled with MAC.

• 17. Wait 5 min for the anticlotting to operate (see Note 15).

• 18. Submerge the colony into a container filled with PAS solution for 30 s.

• 19. Transfer the colony to an empty collection support.

• 20. Blot gently the colony using paper tissues.

• 21. Fill the collection support with 50 mL PAS to completely submerged the colony.

• 22. Retrieve the PAS solution into a container for later use (see Note 16).

• 23. Place the collection support in the microtransfusion setup (Fig. 1c).

• 24. Choose a collection point using the stereomicroscope (Fig. 1d, e, see Note 17).

• 25. Move the needle close to the target collection point using the micromanipulator (see Note 18).

• 26. Empty the needle from the MBE.

• 27. Insert the needle into the tunic towards the vessel lumen until the hemolymph enters the needle (Fig. 1f).

• 28. Apply negative pressure in the needle to compensate for reductions in the flow rate of hemocytes (see Note 19).

• 29. Gently rock forward/backward the needle inside the vessel to prevent hemocytes from attaching to the needle (see Note 20).

• 30. Alternate the release of the pressure with increases in negative pressure for collecting hemolymph (see Note 21).

• 31. Collect as much hemolymph as required (Fig. 1g, see Note 22).

• 32. Release the pressure valve in the microinjector.

• 33. Retract the needle form the vessel.

• 34. Empty the glass micropipette into the previously coated tube.

• 35. Unmount and discard the used glass micropipette.

• 36. Keep this hemolymph at RT until use (see Note 23).

Fig. 2

Manipulating hemocytes. (a) Counting cells on a hemocytometer using Trypan blue to detect dead cells (none visible) with (b) a magnification of the area delimited in (a) where five cells are visible. (c) Measuring the purity of a collection using a linear regression of the absorbance of a purity scale. Absorbance of the reference samples are depicted using gray dots, purity scales as gray lines, the linear regression as a bold black line, and its 95% confidence interval by a light gray surface. Samples used to measure medium (n = 23) and high (n = 18) purity are depicted in blue and red, respectively. Their corresponding average values and standard deviation are shown as a bold line overlapping a light area. R² is the regression coefficient and Δ the slope of the regression. (d) Labeled hemocytes with (e) a magnification of the area delimited in (d) and (f) the corresponding fluorescence of the lipophyllic dye (MemGlow 640) with (g) the corresponding magnification. All scale bars are 100 μm

3.2 Characterization of Hemolymph Collection

Depending on the downstream application, careful characterization of the sample needs to be undertaken. Here we measure the purity of the collection , the amount of collected cells, the viability of these hemocytes and calculate the in vivo cell concentration and hemolymph volume.

• 1. Incubate a P2 tip and a P200 tip in MBE for 5 min (see Note 24).

• 2. Pipette 200 μL of MBE in a 1.5-mL tube.

• 3. Vortex the tube for 5 s to coat its internal surface.

• 4. Wait for 5 min for the coating to take effect.

• 5. Remove the MBE from the tube.

• 6. Pipette 8 μL of MBE in the coated tube.

• 7. Pipette slowly the hemolymph ten times using the emptied coated P200 tip to mix it.

• 8. Transfer 2 μL of hemolymph to the coated tube using the emptied coated P2 tip.

• 9. Add 10 μL of 2× brightfield cell viability dye to the diluted cells.

• 10. Load 10 μL of cell mix in each side of the hematocytometer.

• 11. Wait for 5 min for all cells to settle at the bottom of the chamber.

• 12. Measure the concentration of viable and dead cells in both the wells according to the manufacturer’s instructions (Fig. 2a, b).

• 13. Multiply the obtained average cell concentration by 10 to calculate the concentration of the collected sample (see Note 11).

• 14. Spin the collected hemolymph 12 min at 800 rcf.

• 15. Transfer the supernatant to a new 1.5-mL tube.

• 16. Measure the total amount of supernatant.

• 17. Multiply the determined cell concentration by the volume of supernatant to determine the total number of collected cells.

• 18. Add 20 μL of MBE to the pelleted cells.

• 19. Resuspend the collected hemocytes by pipetting ten times the bottom of the tube using the coated P200 tip (see Note 25).

• 20. Initialize the microvolume spectrophotometer according to the manufacturer’s instructions using MBE as blank.

• 21. Calibrate the microvolume spectrophotometer to measure absorption at 630 nm.

• 22. Measure absorbance of 2 μL of the hemolymph supernatant.

• 23. Measure absorbance of 2 μL of each dilution of the purity scale (see Note 26).

• 24. Repeat steps 21 and 22 two more times.

• 25. Perform a linear regression on the measurements of the absorption of the purity scale.

• 26. Use the regression to infer the purity of the collected hemolymph (Fig. 2c, see Note 13).

• 27. Divide the previously determined cell concentration by the inferred purity to obtain the in vivo cell concentration (see Note 24).

• 28. Multiply the volume of collected supernatant with the inferred purity to obtain the in vivo volume of hemolymph.

• 29. Keep the hemocytes at RT until use (see Note 22), freeze the serum and store at −80 °C (see Note 27).

3.3 Flow Cytometry Analysis of Hemolymph

As described in the introduction, hemolymph has been used for a variety of applications in a number of publications. Here we present our protocol to label hemocytes, analyze them using flow cytometry, and fix them for morphological purposes.

• 1. Transfer the desired amount of cells into a coated 1.5-mL tube (see Note 28).

• 2. Spin the hemocytes for 12 min at 800 rcf.

• 3. Remove the supernatant.

• 4. Resuspend the pellet by gently pipetting using a coated P200 tip in 20 μL of MBE.

• 5. Add 0.1 μL of the live cell labeling dye to the cells.

• 6. Mix by pipetting ten times using a coated P200 tip.

• 7. Wrap the tube in aluminum foil.

• 8. Leave 30 min to incubate at RT.

• 9. Spin the hemocytes for 12 min at 800 rcf.

• 10. Remove the supernatant.

• 11. Resuspend the cells in 47.4 μL of MBE (Fig. 2d–g).

• 12. Add 2.5 μL of the cell viability dye.

• 13. Add 0.1 μL of the DNA live labeling dye.

• 14. Mix by pipetting ten times using a coated P200 tip.

• 15. Keep the tube in aluminum foil at RT.

• 16. Transfer 20 μL of unstained cells into a coated 1.5-mL tube as negative control.

• 17. Pipette the cells through a cell strainer to remove cell aggregates.

• 18. Dilute the calibration beads in the unstained cells according to the manufacturer’s instructions.

• 19. Initialize the flow cytometer according to the manufacturer’s instructions.

• 20. Calibrate the voltage and the gain of each detector using the negative control (see Note 29).

• 21. Run 10 μL of stained cells to calibrate the gain in the fluorescent channels where signal is expressed.

• 22. Gate relevant events as single cells, DNA-positive, and live cells (see Note 30).

• 23. Record 10,000 events of labeled hemocytes (see Note 31).

• 24. Gate subpopulations of hemocytes in the forward vs. side scatter to quantify their relative proportions (see Note 32).

• 25. Add an equal volume of the fixative solution to the leftover cells for morphological fixation.

• 26. Mix by pipetting ten times using a coated P200 tip.

• 27. Incubate the cells for 30 min at 4 °C.

• 28. Spin the hemocytes 12 min at 800 rcf.

• 29. Replace the supernatant with 3.3× K-PBS.

• 30. Resuspend the cells by pipetting ten times using a P200 pipette.

• 31. Store fixed cells at 4 °C, use within 6 months.

3.4 Injection of Compounds and Cells

Given that the vasculature is a closed system, there is a limitation in the volume and rate of injection that can be achieved, which depends on the size of the colony. This protocol typically yields to the injection of 40 μL of solution in 15 min in colonies composed of more than five adults (Fig. 3c, see Notes 33 and 34).

• 1. Follow steps 1–4, Subheading 3.1, to prepare a colony for injection .

• 2. Trim a glass micropipette using a sharp razor blade to obtain a tip diameter of 17–30 μm (see Note 13).

• 3. Load 200 μL of PAS into a cut P200 pipette tip (see Notes 35 and 36).

• 4. Follow steps 7–11, Subheading 3.1, to prepare the injection equipment.

• 5. Transfer the colony to an empty collection support.

• 6. Place the collection support in the microtransfusion setup.

• 7. Choose a collection point using the stereomicroscope (see Note 37).

• 8. Move the needle close to the target collection point using the micromanipulator (see Note 18).

• 9. Insert the needle into the tunic toward the vessel lumen until the hemolymph enters the needle (Fig. 3a, see Note 38).

• 10. Slowly increase the pressure to carefully inject a small amount of the solution (<1 μL).

• 11. Observe the dye spreading through the surrounding vasculature (Fig. 3b, see Note 39).

• 12. Continue increasing the pressure to inject the required amount of solution (Fig. 3c, see Note 33).

• 13. Follow steps 32–35, Subheading 3.1, to remove the micropipette from the colony.

• 14. Leave the colony resting for at least 5 min to allow systemic distribution of the stain through all the vasculature (Fig. 3d, see Note 40).

• 15. Load 20 μL of labeled cells into a trimmed glass micropipette.

• 16. Repeat steps 4–14 to reinject labeled hemocytes (Fig. 3e).

Fig. 3

Microinjections in Botrylloides diegensis. (a) Vascular system with a micropipette inserted in the vessel lumen loaded with PAS. (b) Properly inserted micropipette starting to inject a dyed solution. (c) Average injection rate over time, separated between intravascular (red, n = 5) and subendostylar (blue, n = 5), overlaid with the global average (black). Display are the individual collections (thin), the average collection (thick), and the corresponding standard deviation (light area). (d) Systemic distribution of the injected medium 5 min after the end of the microinjection . (e) Injection of labeled hemocytes (arrows) in the recipient colony. All scale bars are 500 μm

4 Notes

• 1. Alternatively, natural sea water or artificial seawater could replace K-PBS after 0.22-μm sterile filtration. However, we have observed reduced hemocyte clotting in potassium- and magnesium-depleted media.

• 2. Adapt the salt concentration and the pH of the K-PBS to the culturing conditions used for your colonial ascidians. In our lab, Botrylloides diegenesis is cultured at 32 ppt salinity and pH 8.2.

• 3. Although we have used MBE up to 2 days old with success, we have observed that crystals and debris start forming after 6–10 h. We thus advice to prepare this medium fresh every time.

• 4. Brilliant Blue FCF is usually marketed as E133 food dye [38], a non-toxic, inexpensive and hydrophilic dye that contrasts well with the typical orange color of our colonies. Other dyes, and food dyes in particular, can be used instead. Fluorescent dyes could also be used, but phototoxicity during imaging should be carefully monitored.

• 5. Concentration has been empirically determined as the highest amount of dye that permits the visualization of the vessels on our microscope (Leica MS5), other setups might require different concentrations. Different batches of dye may have different initial concentrations.

• 6. The 3D-printed profile accommodates a standard 75 × 25 mm glass slide onto four supports located in each corner of the slide within a disk that fits tightly in a petri dish. These supports allow the convenient handling of colonies, including those with systems growing on the sides and back of the glass slide. Colonies can be maintained submerged and in a fixed position for long durations. Standard triangle language (STL) file of the 3D model is available upon request.

• 7. We have used capillaries with and without filament with similar results. Capillaries without filament have been preferred as they are cheaper.

• 8. The purity of hemolymph is inversely proportional to the percentage of PAS present in the collected samples. We use MBE as substitute to the hemolymph for our calibration scale.

• 9. We have tried Vybrant DiO (Molecular Probes) as alternative live labeling dye. We obtained good labeling and fluorescence, but it induced cell aggregation.

• 10. Important factors for accessibility are the possibility to visualize and identify vessels adequate for puncture, sufficient room around the puncture point to manipulate the needle and an internally soft tunic. In the case of B. schlosseri, the outer tunic is pretty tough, but a small incision using a micro knife gives access to the soft inner tunic.

• 11. On average, we have observed a concentration of 278,000 ± 31,000 (n = 16) viable cells. An almost three times higher cell concentration has been observed in colonies undergoing takeover. No dead cells should be present in a control sample.

• 12. Bigger colonies can yield higher amounts of hemolymph and allow larger injection volumes. However, the accessibility of good collection points is the main factor for good collections and injections.

• 13. Using our capillaries (TW100-6, World Precision Instruments), the diameter of the glass micropipette for the collection of the hemolymph can vary between 17 and 30 μm. Be aware that by using a small diameter (17–20 μm), the collected hemolymph is pure (96%) but the volume will be low (2–5 μL) before the tip clots. A glass micropipette with a larger diameter (20–30 μm) allows to collect a larger volume of hemolymph with a lower grade of purity (67%). Larger is the diameter, higher is the possibility that the PAS surrounding the colony goes inside the micropipette but greater is the amount of hemolymph collected.

• 14. The trimmed tip should fit snugly onto the rear of the capillary. Excess trimming of the P200 will leak MBE, preventing the loading of the capillary. New tip will have to be trimmed. Insufficient trimming will prevent the capillary to enter the tip; additional careful trimming should be done.

• 15. Anticlotting efficiency might vary in different species of colonial ascidians. We have observed an improved efficiency in B. schlosseri compared to B. diegenesis . Longer incubation times can be used to improve the anticlotting effect. We have soaked animals in MAC for up to 30 min without any noticeable deleterious effect.

• 16. Although PAS should be prepared fresh, multiple collections taking place on the same day can use the same medium.

• 17. Favor the following conditions for collection and injection : vessels with a big diameter (longer suction times, less clotting), Y-shaped vessel junctions, vessels embedded in a thick tunic (increases purity by reducing leakage into the glass micropipette).

• 18. Favor the lowest angle possible between the needle and the colony. The objective is that the needle goes through as much tunic as possible before puncturing the vessel to provide natural isolation with the PAS.

• 19. It is easier to properly monitor the flow of hemocytes inside the microneedle than at its tip.

• 20. Movement should be restricted within the vessel to preserve the purity of the sample. Excessive movements might puncture the other side of the vessel or create a gap between the entry site and the needle, thus allowing PAS to enter the vessel.

• 21. Excessive negative pressure will lead to the collapse of the vessel, preventing collection altogether. Vessel will ultimately recover but this is particularly inefficient. Low negative pressure will collect hemocytes at a reduced speed that will allow them to clot in the tightest portion of the needle.

• 22. Select a new collection point when the hemolymph inside the vessels is not circulating any more. This very likely indicates that a clot has formed in a near vascular junction to deviate the circulation to other vessels. If the tip is clogged, try increasing the negative pressure drastically to force it through the pipette. If that does not work, it is necessary to change the pipette.

• 23. Viability tests of hemocytes using Trypan Blue have shown that when kept in MBE, half of the total amount of cells die every 24 h. Storage of hemolymph at 4 °C did not show any significant differences compared to the storage at RT.

• 24. All handling of hemocytes should be done with coated tips. Reusing coated tips as much as possible is our preferred approach. Alternatively, coating multiple tips is possible. To coat multiple tips in parallel, we soak them loaded with MBE in a 50-mL glass beaker filled with MBE.

• 25. Avoid vortexing hemocytes as we have observed increased cell death after such treatment.

• 26. In our setup (NanoDrop One, Thermo Fisher Scientific), absorptions of the reference samples of our purity scale are remarkably constant between different measures made on different days (Fig. 2c). For new samples, we thus measure only a fresh 90% and 30% reference samples to confirm that the previously measured purity scale can be used for these values.

• 27. We have been successfully using the Simultaneous Metabolite, Protein, Lipid Extraction (SIMPLEX) approach [39] to process freeze-dried samples for metabolomic and proteomic analysis.

• 28. Samples from different histocompatible colonies can be pooled to reach the desired amount of cells, if it exceeds the total amount of cells collected in one microtransfusion. Working with subclones of the same colony is even simpler. Note that the blastogenic cycle of isolated subclones can slowly diverge. Make sure that the pooled samples are in the same blastogenic stage if that is relevant for the experiment.

• 29. Hemocytes in Botrylloides have a size ranging between 5 and 35 μm in diameter [12]. Use the calibration beads to determine the minimum limit for the Forward Scatter (FSC). Use the signal of the unstained cells to set the voltage for all detectors. Note that colonial tunicate’s hemocytes do have a significant amount of autofluorescence.

• 30. Although we favor the combination of dyes DRAQ5/Calcein Violet for their quantitativity, DAPI/propidium iodide is a very popular and functional alternative.

• 31. A total of 10,000 events is typically considered as the minimal number of events required to produce a flow cytometry plot. For higher resolution and better analysis, this number should be increased up to 50,000 events. For a quantitative analysis of expression and labeling, multiple controls need to be acquired together with the sample of interest. In particular, a negative control and single-label samples [40].

• 32. In Botryllus schlosseri, seven subpopulations of hemocytes have been gated successfully using the forward vs. side scatter [28]. Specific labeling further subdivided hemocytes into 11 populations.

• 33. The injections can be done either intravascularly or directly into a zooid’s subendostylar sinus. To avoid leakages, rates of injection should not exceed 2 μL/min intravascularly and 5 μL/min into the sinus.

• 34. The maximal value that we have been able to inject in one isolated zooid without leakage is 15 μL. Consequently, even very small system of more than three zooids allow the injection of the 40 μL contained in the glass micropipette.

• 35. Injecting PAS is being used here as an example and has no additional application than transiently highlighting the vasculature. However, we suggest using it as a general marker for injections as injecting transparent solutions is more difficult to monitor and control.

• 36. Loading solutions can be done by several means. Loading using a cut P200 tip is the fastest and cheapest but requires large volumes of solution. Loading using microcapillary pipette tips is pretty fast too, but these tips are relatively expensive. Alternatively, mounting the empty micropipette on the microinjection setup and loading it through its tip by applying negative pressure are slow but inexpensive.

• 37. Good injection points are similar to good collection points (see Note 14), with the addition of the possibility to inject even faster through the subendostylar sinus (see Note 29).

• 38. Make sure there is no hemolymph leaking around the puncture site, otherwise the injected material will mostly leak outside of the vascular system.

• 39. Failure for the solution to diffuse properly is a sign that the needle is not well inserted in the vessel lumen. Most typically, using a new injection point will be necessary to allow a leak-less injection .

• 40. PAS will diffuse through the vessel epithelium and the tunic and will be completely eliminated from the colony in less than 30 min.