DC isolation problem: too many macrophages - (Jan/15/2008 )
I'm currently working with immature dendritic cells, but recently I've been getting a low yield.
The protocol I use is to flush stem cells out of femur bones, usually using 4 mice per experiment I run. Then after lysing RBC and washing in PBS, adding 20ng/mL GM-CSF and 10ng/mL IL-4 to differentiate, and incubating for 4 days in 2 T-75 flasks. Recently it seems like the majority of cells become macrophages and adhere very tightly to the flasks. I don't think I'm doing anything differently than I used to, and I used to get very high numbers of DC. Now I hardly get enough to run my experiments. Anyone have ideas as to what might be favoring macrophage over DC differentiation?
-Andrew L-
QUOTE (Andrew L @ Jan 16 2008, 02:24 AM)
I'm currently working with immature dendritic cells, but recently I've been getting a low yield.
The protocol I use is to flush stem cells out of femur bones, usually using 4 mice per experiment I run. Then after lysing RBC and washing in PBS, adding 20ng/mL GM-CSF and 10ng/mL IL-4 to differentiate, and incubating for 4 days in 2 T-75 flasks. Recently it seems like the majority of cells become macrophages and adhere very tightly to the flasks. I don't think I'm doing anything differently than I used to, and I used to get very high numbers of DC. Now I hardly get enough to run my experiments. Anyone have ideas as to what might be favoring macrophage over DC differentiation?
The protocol I use is to flush stem cells out of femur bones, usually using 4 mice per experiment I run. Then after lysing RBC and washing in PBS, adding 20ng/mL GM-CSF and 10ng/mL IL-4 to differentiate, and incubating for 4 days in 2 T-75 flasks. Recently it seems like the majority of cells become macrophages and adhere very tightly to the flasks. I don't think I'm doing anything differently than I used to, and I used to get very high numbers of DC. Now I hardly get enough to run my experiments. Anyone have ideas as to what might be favoring macrophage over DC differentiation?
Maybe you should use bacteriological dishes?
-Bradley-
QUOTE (Andrew L @ Jan 15 2008, 06:24 PM)
I'm currently working with immature dendritic cells, but recently I've been getting a low yield.
The protocol I use is to flush stem cells out of femur bones, usually using 4 mice per experiment I run. Then after lysing RBC and washing in PBS, adding 20ng/mL GM-CSF and 10ng/mL IL-4 to differentiate, and incubating for 4 days in 2 T-75 flasks. Recently it seems like the majority of cells become macrophages and adhere very tightly to the flasks. I don't think I'm doing anything differently than I used to, and I used to get very high numbers of DC. Now I hardly get enough to run my experiments. Anyone have ideas as to what might be favoring macrophage over DC differentiation?
The protocol I use is to flush stem cells out of femur bones, usually using 4 mice per experiment I run. Then after lysing RBC and washing in PBS, adding 20ng/mL GM-CSF and 10ng/mL IL-4 to differentiate, and incubating for 4 days in 2 T-75 flasks. Recently it seems like the majority of cells become macrophages and adhere very tightly to the flasks. I don't think I'm doing anything differently than I used to, and I used to get very high numbers of DC. Now I hardly get enough to run my experiments. Anyone have ideas as to what might be favoring macrophage over DC differentiation?
In the past when I have isolated BM DCs I cultured the BM (after RBC lysis) in Flt3 and would only look for DCs after about 10 days. I got this protocol from a DC lab. Hope it helps
-Clare-