This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Isolating and culturing dendritic cells (Dendritic Cell J558 Protocol)
Aditya Rayasam
Sandor Lab
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Isolating and culturing dendritic cells
Keywords
BMDC 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!
This is a list of supplementary files associated with this preprint. Click to download.
- J558protocol.docx
J558 DC protocool DC protocol
Version History
CURRENT STATUS: Posted
Version 1
Posted 17 Mar, 2015
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Subject Areas
Biological techniques
Method Article
Isolating and culturing dendritic cells (Dendritic Cell J558 Protocol)
Aditya Rayasam
Sandor Lab
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 17 Mar, 2015
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Isolating and culturing dendritic cells
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!