Botryllus schlosseri Epithelial Tissue Isolation Protocol

This protocol is version 01.25.24

Protocol

Materials

Equipment

• Biosaftey cabinet
• Nikon SMZ745T Stereoscopic Microscope with Excelis camera
• Evos XL Core inverted microscope
• Aspirator
• Spray bottle
• Two 15 cm diameter glass culture dish
• Small shallow dish (size not important)
• Curved forceps
• Pipette wand
• 1000 mL waste beaker
• 1000 uL pipette
• 100 uL pipette

Consumables

Solution preparation: - 0.22 um filter units - Penicillin-Streptomycin (100x stock) - Amphotericin-B (250 ug/mL stock) - Gentamicin (50 mg/mL stock) - 75 ppt filtered seawater (FSW) - Refractometer

During dissection: - BH Supplies Insulin Syringes U-100 28G 1ml/cc 5/16” (8mm) - 100 um cell strainers - 1.5 mL tubes (number dependent on how many samples) - 30% ethanol - Autoclaved 27 ppt artificial seawater (ASW)

During tissue seeding: - Corning 6 well plates or T25 culture flasks - 60 mm petri dishes - 50 mL, 10 mL, and 5 mL serological pipettes - 1000 uL pipette tips - 70% ethanol

Solutions

Concentrated Filtered Seawater (for media formulation)

1. Make concentrated saltwater

Option A: Mix together the below two solutions:

• To 500 mL of MilliQ water
  • 54.68 g NaCl
  • 1.648 g KCl
  • 2.884 g CaCl2·2H2O
• To 500 mL of MilliQ water
  • 22.28 g MgSO4·7H2O
  • 12.2 g MgCl2·6H2O

Option B: Formulate 500 mL of concentrated salt water (75 ppt) solution from Red Sea Salt.

• Verify salinity using refractometer prior to filtering.

2. Filter solution through 0.22 um filter unit.

Artificial Seawater

36.6 grams (g) of Red Sea Salt per liter to make solution at ~30 ppt.

Artificial Seawater - 0.05% Gentamicin (ASW-G)

This solution is essential for the isolation of tunicate individuals 24 hours prior to dissection and during dissections.

In the biosaftey cabinet add together the following:

• 500 mL of artificial seawater (~850 mOsmo/kg)
  • Does not have to be autoclaved or filtered
• 238.8 uL gentamicin (50 mg/mL stock)

Artificial Seawater - 1% Pen/Strep & 0.01% Amp B (ASW-PSA)

This solution is used as the media in which the tunicate is submerged during dissection.

In the biosafety cabinet add together the following:

• 494.5 mL filtered seawater (~850 mOsmo/kg)
• 500 uL Amphotericin B (250ug/mL stock solution)
• 5 mL of Penicillin-Streptomycin (100x)

Tunicate Culture Medium (TCM) with 1.4% Pen-Strep and 0.1% Amp B

In the biosafety cabinet add together:

• 45.6 mL of L-15 media
• 21.7 mL of FSW (to adjust media milli-osmolality from ~300 mOsmo/kg to ~850 mOsmo/kg)
• 2.4 mL fetal bovine serum (FBS)
• 1 mL HEPES (20 mMol/L)
• 1 mL Pen/Strep (100x)
• 100 uL Amphotericin B (250 ug/mL)

Cell Isolation Procedure

Be sure to have sufficient stocks of the above three solutions prior to following the below procedures.

1. The day prior to bud isolation

• Select an individual (one glass slide) with at least 30 zooids completely adhered to the slide. Health score must be 7 and above. Target stages C1-D. Earlier stages do not produce monolayers as frequently.

• Remove individuals from recirculating seawater system

• Use a razor blade to clean the glass slide

• Spray animal over sink with 70% ethanol for 5 seconds or until glass slide is completely coated with ethanol

• Rinse animal with autocalved artificial seawater until no ethanol is visible.

• Place animal in 15 cm glass petri dish containing 100 mL of ASW-G.

• Parafilm and perforate petri dish.

• Leave overnight for starvation on lab bench at room temperature.

2. Tissue Dissection

• Remove colony from ASW-G.

• Wash colony and substrate for 10 seconds with 30% ethanol.

• Rinse colony with autoclaved artificial seawater

• Immerse colony in 15 cm diameter, glass dish filled with ~60 mL of ASW-PSA. For each new colony microdissection, rinse the 15 mL glass plate with 70% ethanol in triplicate, followed by triplicate rinse of ASW-PSA prior to refilling plate with more ASW-PSA.

• Under stereomicroscope on lab bench take picture of entire colony (all tissue to be dissected with Excelis camera.

• Record blastogenic stage, zooid or primary bud number, and health score.

• Using a pair of 28G syringe needles, peel open the tunic and remove primary buds.

• Every time a primary bud is removed, place it in cell strainer that itself is also immersed in the same 20 mL of ASW-G. Use a clicker to keep track of the number of primary bud you collected.

• Once you have collected a group of 10 primary buds in the cell strainer, rinse all primary buds for 5 seconds with 30% ethanol followed by a 5 second rinse with autoclaved artificial seawater.

• Use same pair of insulin needles to transfer over the primary buds to the 1.5 mL tube containing 1 mL of TCM. This can be done by placing the cell strainer in ASW-PSA under the stereomicroscope and carefully rolling the primary buds onto one of the needles. Make sure not to burst them.

• Transfer all tubes containing bud tissue to the biosaftey cabinet in TPS for cell seeding.

3. Seeding tissue and cell maintenance

• In the biosaftey cabinet, use a 1000 uL pipette tip and aspirate into the solution to get the buds back into suspension and transfer to plastic 60 mm petri dish.

• Using the same 1000 uL pipette tip transfer buds one at a time to a fresh culturing dish. Make sure to transfer only a small amount of liquid (about a drop) per bud.

• Desiccate tissue slightly to ensure adherence to the substrate.

• Add 0.5-1 mL of TCM to each well. Be sure to not touch the tip of the pipette to the wells and to have the lowest flow possible when adding in media to not agitate the tissue and have it unadhere from plate.

• Incubate cells at 18 C, 91% humidity.

• Replace 100% of the TCM every 2-3 days for plates and once a week for flasks.