Developmental Biology Immunology Cell Culture

scientificprotocols authored almost 3 years ago

Authors: Ivan Zanoni, Renato Ostuni & Francesca Granucci

Abstract

Protocol for generating mouse dendritic cells from bone-marrow progenitor cells used in our Nature paper.

Reagents

  1. GM-CSF-transduced B16 cell line.
  2. BMDCs culture medium recipe (conditioned medium):
  3. HI FBS (EuroClone) – 10%
  4. L-Gln (EuroClone) – 2mM
  5. Penicillin/Streptomycin (EuroClone) – 50 U/ml
  6. Beta-mercaptoethanol (EuroClone) – 50 microM
  7. B16-GMCSF growth supernatant – 10%
  8. IMDM (EuroClone) – to volume

Procedure

  1. Flush mouse tibiae and femurs with ice-cold PBS through a 70 μm-wide cut-off cell strainer.
  2. Centrifuge 5’ at 1400 rpm. Resuspend pelleted cells in conditioned medium (supplemented with 10% of growth supernatant of GM-CSF-transduced B16 cells).
  3. Seed 7×10e6 cells in 100×20 mm non-treated cell culture plates in 10 ml of conditioned medium.
  4. Incubate at 37 °C – 5% CO2.
  5. On day 4 and 7 add 5 ml of pre-warmed conditioned medium.
  6. At day 8/9 the percentage of CD11c+ cells should be higher than 90% as measured by FACS analysis. BMDCs are then ready for experimental use.
  7. Harvest the supernatant and gently wash the plate once with 5 ml of pre-warmed PBS.
  8. Incubate 2’ with 5 ml of 2mM EDTA at 37 °C – 5% CO2.
  9. Collect cells, wash once with PBS.
  10. Centrifuge 5’ at 1200 rpm and resuspend pelleted cells in conditioned medium.

Associated Publications

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation, Ivan Zanoni, Renato Ostuni, Giusy Capuano, Maddalena Collini, Michele Caccia, Antonella Ellena Ronchi, Marcella Rocchetti, Francesca Mingozzi, Maria Foti, Giuseppe Chirico, Barbara Costa, Antonio Zaza, Paola Ricciardi-Castagnoli, and Francesca Granucci, Nature 460 (7252) 264 - 268 09/07/2009 doi:10.1038/nature08118

Author information

Ivan Zanoni, Renato Ostuni & Francesca Granucci, University of Milano-Bicocca

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.137. Origginally published online 18 June 2009.

Average rating 0 ratings