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Uptake of dead cells by D2SC1/Raw/Flt3-DC/GM-DC
Laboratory protocol
Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma
Staining of GM-DC/Raw with anti-CD11c antibodies
Coculture of DCs and cells to be taken up (1(-2)x105 + 1(-2)x105 cells in 12-well) for 4 h to 24 h at 37°C
Take supernatant and freeze at -80°C for analysis of DC-stimulation
Trypsinize cells have and fix in 4 % PFA (or formalin)
FACS analysis
Protocol Steps:
-
Staining of D2SC1/Flt3-DC and of the cells to be taken up
Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma
-
Staining of GM-DC/Raw
Staining of GM-DC/Raw with anti-CD11c antibodies
-
Coculture
Coculture of DCs and cells to be taken up (1(-2)x10^5 + 1(-2)x10^5 cells in 12-well) for 4 h to 24 h at 37°C
-
Supernatant freezing
Take supernatant and freeze at -80°C for analysis of DC-stimulation
-
Trypsinize
Trypsinize cells and fix in 4 % PFA (or formalin)
-
FACS analysis
created over 16 years ago
(2 March 2009)
last modified over 13 years ago
(28 September 2011)
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