Culture of MutuDC cell line

Culture of MutuDC (mouse cDC1 line)

Cell line: MutuDC 1940

Culture Media:

500ml IMDM (Gibco, cat no:12440053)

50ml DC grade Heat Inactivated-FCS (Please note: our FCS is batch tested for DC culture) (In vitro technologies)

5ml Pen/Strep (Sigma, cat no: P0781)

0.25ml 2-ME (Gibco, cat no: 21985023, stock at 55mM, 100x)

Adjust Osmolarity to 308 (± 2) mOsmo/kg using 1 M NaCl 

Buffers:

Dulbecco PBS (DPBS) (Gibco, cat no 14190250). Adjust osmolarity to 308 mOsmo/kg using 1M NaCl.

5mM EDTA in DPBS (308 mOsm/kg)

MutuDC culture protocol

1. MutuDC are only semi-adherent.
2. Collect cell culture supernatant from cell culture flask/plate to a tube.
3. To dislodge remaining adherent cells from cell culture flask/plate, add 5mM EDTA, incubate for 1-2min at 37C.
4. Tap flask/use a sterile pipette to detach cells.
5. Use collected Mutu DC culture supernatant (containing MutuDC from step 2) to rinse flask/plate and pool together.
6. Spin cells down at 700 x g for 7min, 4C.
7. Discard supernatant.
8. Resuspend cells with warmed culture media.
9. Passage at 1:3 to 1:5 dilution, every 2-3 days. The cells appear healthiest when grown up to 70% confluence as a monolayer. Please do not allow them to overgrow / grow on top of each other.