Preparation of DCs

Preparation of Dendritic Cells

Step 1. Remove dMLNs from mice. Dendritic cells were harvested from the dMLNs of ~5-10, 3-5 week old Ly5.1 Foxp3^IRES-GFP mice in our colony for ex vivo and FACS experiments, and from cDC deficient mice for qPCR experiments. 

Step 2. Dissociate dMLNs.  5 dMLNs were pooled per 15 ml conical tube (Thermo Fisher #14-959-53A) containing 10 ml RPMI (Thermo Fisher #SH3025502) with 5% bovine calf serum (Thermo Fisher #SH3062602), 1x penicillin/streptomycin (Cytiva HyClone #SV30010), 1 mM sodium pyruvate (Lonza #BW13-115E), 1x non-essential amino acids (Cytiva HyClone #SH3023801), 50 mM beta-mercaptoethanol (VWR #97064-588), 65.8 mg/ml collagenase VIII (Sigma #C2139), and 0.2 U/ml dispase (Thermo Fisher #CB-40235), for 45 minutes at 37°C with 300 RPM continuous magnetic stirring (stir bar 7.9 mm x 12 mm: VWR #58948-116) until tissue was completely dissociated. Suspensions were filtered through an 80 mm mesh filter (Component Supply #U-CMN-80) into a new 15 ml conical tube and centrifuged at 340 RCF for 5 minutes at 4°C.

Step 3. Block and stain cells. The supernatant was removed, and cells were resuspended in 2.5 ml of “sort buffer” PBS (Cytiva HyClone #SH30028.03) containing 5% bovine calf serum and 10 mg/ml anti-CD16/CD32 (BioXCell #BE0307). Cells were incubated on ice for 20 minutes after which 2.5 ml 2x fluorescently-labeled antibody mix was added to each tube (2.5 ml sort buffer with anti-CD3e, anti-B220, anti-CD19, anti-IAb, anti-CD11c, anti-CD103, anti-CD11b; refer to Key resources table for catalog numbers and dilutions). Cells were incubated on ice, in the dark for 30 minutes. Cells were spun at 340 RCF for 5 minutes and washed with 10 ml sort buffer. 

Step 4. Sort cDC subsets. The supernatant was removed, cells were resuspended in 1 ml sort buffer, and cell suspension was filtered through a 40 mm mesh (Component Supply #U-CMN-40) into a sterile 5 ml culture tube with lid (VWR #60818-500). Cells were sorted on a BD FACSAria IIu at no greater than 14,000 events/second into sterile 5 ml culture tubes containing 4 ml complete DMEM (DMEM (Thermo Fisher #SH3008101) with 10% fetal bovine serum (Thermo Fisher #16000044), 1x Glutamax (Thermo Fisher #35050061), 50 mM beta-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES (Thermo Fisher #15630080), 1x non-essential amino acids, and 1x penicillin/streptomycin) using the following markers: migratory cDCs (CD3e^– B220^– CD19^– IAb^hiCD11c^int), resident cDCs (CD3e^– B220^– CD19^– IAb^int CD11c^hi), CD103⁺ SP (CD3e^– B220^– CD19^– IAb^hi CD11c^intCD103⁺ CD11b^–), DP (CD3e^– B220^– CD19^– IAb^hi CD11c^int CD103⁺ CD11b⁺), and CD11b⁺ SP (CD3e^– B220^–CD19^– IAb^hi CD11c^int CD103^– CD11b⁺). 

Step 5. Prepare cDCs for culture. Sorted cells were spun at 340 RCF for 5 minutes and resuspended in 1 ml of complete DMEM. Cells were allowed to rest on ice for 30 minutes, after which cell concentrations were calculated by hemocytometer for culture. 

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