1. Materials
   • Timed pregnant mice at E13-E13.5 for isolating embryonic salivary glands
   • 70% ethanol spray bottle
   • Standard forceps (e.g. Fine Science Tools, 11000-12)
   • Fine sharp scissors (Fine Science Tools, 14060-10)
   • DMEM/F-12 (e.g., Thermo Fisher, 11039047)
   • 100x PenStrep (10,000 units/mL penicillin, 10,000 µg/mL streptomycin; e.g., Thermo Fisher, 15140163)
   • DMEM/F-12 + 1x PenStrep (referred to as “medium” throughout this protocol): add 5 mL 100x PenStrep to 500 mL DMEM/F-12
   • Dissecting microscope (e.g., Leica M80; any common dissecting microscopes should work)
   • Dumont #5 forceps (Fine Science Tools, 11251-20)
   • Dumont #5 fine forceps (Fine Science Tools, 11254-20)
   • Scalpel (Fine Science Tools, 10011-00 and 10003-12)
   • 10-cm dish (e.g., Corning 430167)
   • 35-mm dish (e.g., Corning, 430165)
   • Pyrex spot plate (Fisher Scientific 13-748B)
   • 22x22 mm coverslip (e.g., MilliporeSigma, C9802)
   • Dispase (Thermo Fisher, 17105041); dilute in DMEM/F-12 medium at 5-10 U/mL, aliquot and store at -20°C
   • 5% BSA (MilliporeSigma, A8577; diluted in DMEM/F-12)
   • 200 µL low-retention pipette tips (Rainin, 30389187)
   • 20 µL low-retention tips (Rainin, 30389190)
   • 20 µg/mL FGF7 (R&D Systems, 5028-KG-025); divide into 25 µL aliquots and store at -20°C
   • 100x ITS supplement (Thermo Fisher, 41400045)
   • Growth factor-reduced Matrigel (Corning, 356231; stock is usually 9-10 mg/mL)
   • Ultra-low attachment 96-well V-bottom plate (S-bio, MS-9096VZ)
2. Thaw sufficient amount of Matrigel on ice or in a fridge before dissection.
   • Each epithelial bud will need 5 µL Matrigel stock for culture. Adjust amount based on your needs.
   • Matrigel stock has to be thawed at low temperature to prevent premature solidification, and it can take several hours to completely thaw.
3. Please follow this protocol to isolate intact salivary glands.
   • To make sure all epithelial buds come from submandibular glands, remove the attached sublingual gland right after isolating each gland.
4. Isolate salivary gland epithelial buds by dispase treatment and trituration in a 9-well Pyrex spot plate.
   • Prepare enough volume of 2 U/mL dispase solution by diluting the stock with medium.
     • Usually 6-8 intact glands are treated in one well with 150 µL dispase solution.
     • The actual activity of dispase can vary quite a bit. I have used 0.5 U - 2 U/mL for different batches of dispase. Thus, test each batch.
   • Use forceps to transfer 6-8 intact glands into each well with dispase solution in the Pyrex spot plate.
   • Cover each well with a 22x22 mm coverslip to prevent evaporation.
   • Incubate for 15 min at 37°C.
   • For each well, replace dispase solution with 150 µL 5% BSA for twice to quench dispase activity.
   • Isolate single epithelial buds by trituration using 200 µL low-retention tips (P200 set at 100 µL).
     • At this point, mesenchymal cells will dissociate into single cells, while the epithelial buds and ducts will remain largely intact.
   • Rinse epithelial buds twice by transferring them sequentially into new wells with 150 µL 5% BSA using 20 µL low-retention tips. Cover the wells not being used with 22x22 mm coverslips during the transfers and washes.
   • Keep epithelial buds in 5% BSA while preparing for the next steps.
5. Culture salivary gland epithelial buds in an ultra-low attachment 96-well V-bottom plate.
   • Use a multichannel pipette to fill all edge wells of the 96-well plate with 150 µL medium to prevent evaporation. These include: A1-H1, A12-H12, A2-A11 and H2-H11.
   • Use a multichannel pipette to fill all wells for bud cultures with 47 µL medium to allow for 3 µL carryover volume when transferring the bud.
   • Wash isolated epithelial buds twice in medium without BSA. Use 20 µL low-retention tips (cut for larger opening) to transfer buds.
   • Use 20 µL low-retention tips (cut for larger opening) to transfer one bud into each well for culture with 3 µL carryover volume.
   • Prepare 2x culture mix (assuming 20 bud cultures). FGF7 aliquot is quick to thaw in hand or on the bench.

   Stock	µL for one well	21x (make 1 extra)
   Medium	43	903
   FGF7 (20 µg/mL, 100x)	1	21
   ITS (100x)	1	21
   Matrigel (~10 mg/mL)	5	105

   • To each culture well, add 50 µL 2x culture mix prepared above.
   • Incubate the plate at 37°C with 5% CO₂.