RBC lysis buffer

Sigma

 

Wash media

RPMI (Gibco 21870076)

5% serum (Fisher defined fetal bovine serum SH30070.03)

2% kanamycin

 

DC media

RPMI (Gibco 21870076)

10% serum

1% NEAA 1% Na pyruvate

1% glutamax  

1% kanamycin

50uM Beta mercaptoethanol 

 

hFLT3L stock

R&D systems 10ug/ml

 

GMCSF stock

Preprotech 20ug/ml

 

Antibodies

CD11b, CD11c, B220 (available from a number of sources)

Harvesting bone marrow cells

1. Clean hair and muscle off hind legs of mice. Cut tibia and femur bones out as close to joints as possible. Flush marrow from femur and tibia with wash media using a 10ml syringe attached to a 26 gauge needle. Flush bones from both ends.

 

2. Spin 1500 rpm for 5 minutes.

 

3. Resuspend in 1ml RBC lysis buffer, break up clumps using p1000 pipetteman

 

4. Let stand at room temperature for 10 minutes.

 

5. Wash through a cell strainer using 14 ml wash media.

 

6. Count cells using a cell counter.

 

7. Spin cells at 1500 rpm for 5 minutes.

 

8. Resuspend in DC media for plating.

 

Culturing DCs

9a. Plate PDC at 3-5 x 10⁶ cell/ml using hFLT3L at 100 ng/ml. Culture 3mls of volume in 6 well plates. Culture 9-10 days, do not disturb culture or add media during culture. 

 

9b. Plate DC at 1.5-3 x 10⁵ cells/ml using GMCSF at 20ng/ml. Culture 3 mls of volume in 6 well plates. Culture 5-7 days. If media turns yellow, add more media and GMCSF.

 

11. Harvest floating cells and loosely adherent cells by vigorous pipetting.  Cultures should be analyzed by FACS before use to ensure proper development. For same day experimentation, use cultures on day 7 (DC) or day 10 (PDC). For experimentation involving overnight incubation, use cultures on day 6 (DC) or day 9 (PDC). 

 

12a. For FACS, stain FLT3L cultures with CD11c, CD11b, and B220. Most cells should be CD11c^hi. Two main populations are present within the CD11c^hi gate: one that is B220^lo and CD11b^hi and the second is CD11b^lo, and B220^hi. PDCs are the population CD11c^hi, CD11b^lo, B220^hi. The optimal day to FACS is day 10.

 

12b. For FACS, stain GMCSF cultures with CD11b and CD11c. Most cells should be CD11b positive. The cells that are also CD11c positive are considered conventional DC (CD11b⁺CD11c⁺). The optimal day to FACS is day 7.

 

13. For analysis of cytokine production, cells should be stimulated overnight (16-18 hours). Stimuli may be added directly to cells in 6 well plates, or harvested as above (with or without FACS sorting or MACS enrichment) and replated in the same or new media. 

The use of the indicated RPMI is essential for DC/PDC to develop and should not be altered. Many serums may work well, but use of a particular brand and lot should be tested. PDC cultures should not be disturbed until use, even if media turns yellow.   PDC/DC cultures should be analyzed by FACS prior to use to ensure proper development, as percentage of desired cells may vary greatly.   For experimentation, bulk cultures or sorted/enriched cultures may be used depending on needs.