Using the DMEM no glucose and antibiotic-antimycotics

Prepare CaFSW

Add the follow to 1l of deionized H2O and filter with 0.2µm keep stock in 4C and worm to 25C before using

chemical	gram
NaCl	23g
KCL	0.763g
MgSO4-7H2O	1.89g
MgCl2-6H2O	10.45g
Na2SO4	3g
NaHCO3	0.25g
SrCl2	0.026g

DMEM culture medium

Add to DMEM no glucose (https://www.thermofisher.com/il/en/home/technical-resources/media-formulation.49.html) the follow

|chemical|gram|comment| |NaCl|9.05g| |KCL|0.7g| |CaCl2 2 H2O|0.71g| |MgCl2-6H2O|5.1gg | |Na2SO4|0.5g| |taurine|0.5ml|from stock 1.04µg/20ml DDW| |NaOH3-|1.85g|only if necessary, if the DMEM bought is without NaOH3| |Hepes|2.98g| Keep stock in -20C

Working solution

Dilute the stock DMEM culture medium to 12.5%(vol/vol) in ASW: 20ml DMEM + 140ml ASW

Add to the diluted DMEM mix :

• 1.25% FCS
• 1% antibiotic-antimycotics (1.6ml)
• 5mM glucose (For DMEM without glucose)
• 20 µg/ml aspartic acid (0.0032g)
• 50 µg/ml ascorbic acid (0.008g)
• 1:100 L-Glutamine (1.6ml)
• Final pH 8 – monitor gently with NaOH 1M or NaCl 1M
• Filter all the medium in 0.2µm

Tissue culture preperation

All work must be done in the biological/laminar flow hood with filtered medium All tips should be stored in the hood. Tips, pipettes, etc. should be exposed to UV light for at least 10 minutes

All medium need to be in 26C

Work with 6wells or 5ml petri-dish

• Wash 0.2-0.7 cm long nubbins or fragments of nubbins for 15 min in PVP-Iodine
• 2 min wash in FASW and 1 min in DDW.
• Repeat this step if necessary (this steps are done in order to get rid of all the ciliates in medium)
• Pre Incubate nubbins for 4.5 h in CaFSW +3% antibiotics (slow rpm in room temp)
• Transfer the coral nubbin to culture medium (26C)
• Incubate in humidified chamber/incubator with 12/12 h light dark cycle at 26C for 48h.
• The tissue might fall of the skeleton and if not take it off by gentle pipetation. Take out the skeleton homogenize solution and add a drop of PVP-Iodine for 15 min.
• Transfer the medium to 15ml tube and spin down the medium contains the cells (5 min 500g in RT).
• Change the medium with fresh one
• Filter (0.2µm) the medium with the cells to a sterile plate (1ml to each plate) and add 4ml of fresh medium
• Incubate in humidified chamber with 12/12 h light dark cycle at 26C and change medium every 3 days.