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Aditya Rayasam

Isolating and culturing dendritic cells \(Dendritic Cell J558 Protocol)

see attachment \(Images and Attachments) for a Word document version of this protocol.





J558 plasmacytoma cells = X63-Ag8 transfected with the cDNA of mouse GM-CSF\(produced by PCR), cloned into the expression vector BCMGSNeo \(Karasuyama et al., 1990, JEM 172, 969). These cells are DESCRIBED IN Zal et al., JEM 1994, 180:2089).





The cells are cultured in IMDM supplemented with glutamine / penicillin / streptomycin/ B mercapto \(same DC), 5% FCS \(same batch as the culture of DC). The selection medium to maintain the expression of GM-CSF contains 1mg/ml G418.








To make a large stock of ampoules of frozen cells:


Expand cells in selection medium \(they should never exceed 1.5 – 2 x106)


Wash 2x in medium without G418


Freeze at 5 x 10 ^ 6 / 90% FCS ampoule \(same batch as the culture medium) -10% DMSO





To produce conditioned medium:


Thaw a vial by washing in medium without G418 1x


Inoculate a 175cm2 flask in 50 ml of medium with 5% FCS without G418


When there are enough cells \(about 3 days), seed them into 500 ml of medium with 5%FCS without G418, with 2x105 cells / ml \(in "spinner" of 500ml)


3 days later, collect the conditioned medium:





Centrifuge culture 5 min at 1300 rpm


Filter the supernatant through 0.22 um





Re-seed cells at 2x105 cells / ml in approximately 3 L of medium with 5% FCS without G418 \(we use a 3L spinner at this stage)





3 days later, collect the conditioned medium as above


We can rebuild a 3-day conditioned medium with the cells recovered but not more \(discard cells then)





The conditioned medium is kept at 4 C, maximum 4 months, and kept sterile 


For longer storage you can aliquot and freeze at -80C


In ELISA, the concentration of GM-CSF should be more than 20ng/mL


The filtration of the supernatant repeatedly can cause it to lose a part of its activity. Please do not filter more than once the supernatant obtained.


Complete Iscove’s Modified Eagle's Medium \(IMEM)


 5mL Pen/Strep


 5mL L-glut     


0.5mL -MCE


 50mL \(10% \(5% ok too) Heat inactivated Special DC FCS \(South American  Origin\!\!, \(Tested to be the best for DCs)


 50mL GM-CSF


 +389.5 IMDM


Frozen FBS stocks \(special DC FBS), L-glut, P/S in freezer by back elevator in drawer. Use Ike’s frozen FBS aliquots. Stock 500mL FBS is already heat inactivated\!

CC BY-NC 3.0

Isolating and culturing dendritic cells (Dendritic Cell J558 Protocol)

Biological techniques

Method Article

Aditya Rayasam

Aditya Rayasam

Sandor Lab

Corresponding Author

DOI:

10.1038/protex.2015.024

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BMDC protocol

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Sandor Lab

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DOI:

10.1038/protex.2015.024

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