Generation of FAM150A-conditioned and FAM150B-conditioned media

This protocol is used to generate FAM150A and FAM150B conditioned medium if you don't have purified ligand proteins. The plasmids used are pTT5-FAM150A-HA and pcDNA3-FAM150B-HA (both with HA tag at C terminal, for detailed sequence please see the end of this protocol).

1) Seed HEK293 cells (~5x10⁶cell/dish) to 10-cm dishes in DMEM complete medium;

2) When the cells reach 80-90% confluence (monolayer), transfect with 8ug of pcDNA3 empty vector (control) or pTT5FAM150A-HA or pcDNA3-FAM150B-HA plasmid using Lipofectamine 2000 or Lipofectamine 3000 (less toxicity) according to manufacturer's recommendation;

3) Six hours after transfection, change the medium to fresh medium without serum;

4) Further culture the cells for another 48 hours and then collect the medium;

5) Clarify the medium by centrifugation at 2,000rpm for 5 minutes to remove cells and cell debris;

6) Keep medium at 4C fridge (can last for two weeks) or aliquote and freeze at -20C.

pTT5-FAM150A-HA (modified based on pTT5-FAM150A, a generous gift from Five Prime Therapeutics), the sequence of FAM150A-HA is shown below (Yellow: FAM150A CDS, green: linker, cyan: HA tag, red letters: start and stop codons)

ATGCGGCCCC TTAAGCCCGG CGCCCCTTTG CCCGCACTCT TCCTGCTGGC GCTGGCTTTG TCCCCGCACG GAGCCCACGG GAGGCCCCGG GGGCGCAGGG GAGCGCGCGT CACGGATAAG GAGCCCAAGC CGTTGCTTTT CCTCCCCGCG GCCGGGGCCG GCCGGACTCC CAGCGGCTCC CGGAGCGCAG AAATATTCCC AAGAGACTCT AACTTAAAAG ACAAATTCAT AAAGCATTTC ACAGGGCCGG TCACATTTTC ACCAGAATGC AGCAAACATT TCCACCGACT CTATTACAAT ACCAGGGAGT GCTCAACGCC AGCTTATTAC AAAAGATGTG CTAGATTGTT AACAAGATTA GCAGTGAGTC CACTGTGCTC CCAGACCACG CGTTACCCAT ACGATGTTCC AGATTACGCT GTTTAA

pcDNA3-FAM150B-HA was synthesized by the Palmer lab, the sequence of FAM150B-HA with flanked HindIII and NotI sites is shown below (green: restriction enzyme sites, yellow: FAM150B CDS, cyan: HA tag, red letters: start and stop codons)

AAGCTTCACC ATGCGCGGAC CCGGGCACCC CCTCCTCCTG GGGCTGCTGC TGGTGCTGGG GGCGGCGGGG CGCGGCCGGG GGGGCGCGGA GCCCCGGGAG CCGGCGGACG GACAGGCGCT GCTGCGGCTG GTGGTGGAAC TCGTCCAGGA GCTGCGGAAG CACCACTCGG CGGAGCACAA GGGCCTGCAG CTCCTCGGGC GGGACTGCGC CCTGGGCCGC GCGGAGGCGG CGGGGCTGGG GCCTTCGCCG GAGCAGCGAG TGGAAATTGT TCCTCGAGAT CTGAGGATGA AGGACAAGTT TCTAAAACAC CTTACAGGCC CTCTTTATTT TAGTCCAAAG TGCAGCAAAC ACTTCCATAG ACTTTATCAC AACACCAGAG ACTGCACCAT TCCTGCATAC TATAAAAGAT GCGCCAGGCT TCTTACCCGG CTGGCTGTCA GTCCAGTGTG CATGGAGGAT AAGCAGTACC CATACGATGT TCCAGATTAC GCTTAGCGGC CGC

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