ISOLATION SPLENIC DCs (NEGATIVE SELECTION)

Reagents:

Collagenase A (Sigma #11088793001)

DNAse I (Roche #10104159001)

Biotin anti-mouse Ly-6C (Biolegend #128004)

Biotin anti-mouse Ly-6G [1A8] (Biolegend #127604)

Biotin anti-mouse CD3e [145-2C11] (Biolegend #100304)

Biotin anti-mouse CD49b (Biolegend #108904)

Biotin anti-mouse Ter119 (Biolegend #116204)

Biotin anti-mouse/human CD45R/B220 [RA3-6B2] (Biolegend #103204)

MojoSort Streptavidin Nanobeads (Biolegend #480016)

Protocol:

Digest 2 spleens with 1 mg/ml Collagenase A and 50 U/ml DNase I for 30-45 min at 32-35 °C.
Filter though 70 um. Spin down cells at 300 g (10 min).
Resuspend cells in 2.15 ml. (150µl remaining will be for pre-sort control)
Count cells and divide to 1x10⁷cells in 100 µl FACs buffer. (Around 2 ml)
Add Fc block (1/500 dilution) and incubate 10 min on Ice.
Mix biotin conjugated antibodies - 1.5 µl Ly6C (skip for FvBN mice), 2 µl Ter119, 2 µl CD49b, 3 µl Ly6G and 4 µl of: CD3e and B220) in 100 µl total volume of FACs buffer. Depending on the number of total cells this is normally 40 µl Ter119 and CD49b, 60µl Ly6G and 80 µl CD3e and B220 in 1600 µl FACs buffer)
Spin down cells at 300 g (5 min). Add biotin antibody mixture to splenocytes. Mix well. Incubate 20 min on ice.
Spin down at 300 g (5 min). Wash 1x with 2 ml FACs buffer.
Vortex Spheres (MojoSort Nanopartciles). Add 10 µl to 90 ul FACs buffer. Depending on the number of total cells, this is normally: 200 µl beads in 1800 µl FACs buffer.
Add pre-mixed MojoSort beads to splenocytes. Mix well. Incubate 10 min on ice.
Add FACS buffer to 2.5 mls total. Place in magnet for 3 min.
Decant supernatant containing dendritic cells. Count cells and proceed to plate cells.

Tips:

Add 1 µl/ml of EDTA in the last 5 minutes of digestion
When filtering cells, place 5 ml in the 50 ml flask before filtering the cells to reduce the impact.
When decanting cells prioritize speed over getting every cell in order to maximize purity of the DCs.

ISOLATION SPLENIC DCs (POSITIVE SELECTION)