Authors: Aditya Rayasam

### Abstract

Isolating and culturing dendritic cells

### Procedure

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 – 2×106)
Wash 2x in medium without G418
Freeze at 5×10 ^ 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 2×10^5 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 2×10^5 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! 

### Figures 

**J558 DC protocool : DC protocol**

[Download J558 DC protocol](http://www.nature.com/protocolexchange/system/uploads/3565/original/J558_protocol.docx?1424111453) 

### Author information 

**Aditya Rayasam**, Sandor Lab

Correspondence to: Aditya Rayasam (rayasam@wisc.edu)


*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/3723#/procedure) (2015) doi:10.1038/protex.2015.024. Originally published online 17 March 2015*.