Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Isolation of Tumor-Associated Monocyte-Derived Dendritic Cells

1. In a laminar flow hood, remove tumors from CO₂ euthanized mice and place tumors in RPMI medium without fetal bovine serum (FBS).  
   NOTE: Shaving the mice and spraying them with 70% ethanol before removing the tumors is highly recommended to minimize potential contaminations from the fur. Be sure that the tumor does not exceed 25 - 40 mm² since larger tumors have fewer dendritic cells (DC) that tend to be fragile and prone to undergo cell death. If larger numbers of DC are needed, each mouse can be injected with tumor cells at multiple sites.
2. Under a sterile laminar hood, chop the tumors into small pieces (approximately 1 x 1 mm²) using surgery scissors.
   1. Add all tumor fragments from one mouse into a 30-mL sterile flat-bottom tube with magnetic stir bars containing 5 mL of Hank's balanced salt solution (HBSS), 2 mg/mL collagenase IV, and 0.01 mg/mL DNase I.
      CAUTION: Myeloid cells produce energy through glycosylation, even under a steady state. Therefore, only use media that contains glucose (e.g., HBSS, Roswell Park Memorial Institute (RPMI), and Dulbecco's Modified Eagle Medium (DMEM)), as glucose starvation for as little as 10 - 15 min will result in cell death and apoptosis within 24 h. Use only a low endotoxin collagenase IV, as collagenase is produced by Gram-positive bacteria (Clostridium histolyticum).
3. Stir at about 200 - 400 rpm in a 37 ^oC incubator with a magnetic stirrer for 20-30 min.
4. Add 5 mL of complete media and re-suspend vigorously.
5. Filter cells through a 70-µm cell strainer. Centrifuge at 4 ^oC 400 rcf for 5 - 10 min to pellet the cells.     
   CAUTION: Do not attempt to isolate the cells directly following enzymatic digestion, as some tumors are necrotic and contain relatively large amounts of cell debris or extracellular matrix. Apply cells on a 15% density gradient medium to obtain only the cells.
6. Suspend 9 mL of density gradient medium with 1 mL of 10x phosphate-buffered saline (PBS) to adjust osmolality and to obtain 100% Percoll stock solution. Mix 1.5 mL density gradient medium stock solution with 8.5 mL HBSS and mix it vigorously with tumor-derived cell pellet. Gently layer 2 mL of DMEM on the top of a 15% density gradient medium and centrifuge at 400 rcf for 20 min at room temperature. Discard the supernatants, as the cells will form a pellet at the bottom of the tube.
   1. Wash the pelleted cells twice by re-suspending them in 10 mL HBSS containing 2% FBS, 1% penicillin/streptomycin, and 10 mM Ethylenediaminetetraacetic acid (EDTA) (Isolation Buffer), and centrifuge at 4 ^oC 400 rcf for 5-10 min to pellet the cells.
   2. Count cells on a hemocytometer using trypan blue under a light microscope.
7. Re-suspend 1 x 10⁸ cells in 1 mL isolation buffer and incubate them at 4 ^oC with 30 μL of CD11b⁺ conjugated magnetic beads for 15 min.       
   CAUTION: Avoid the use of CD11c-conjugated beads, as this will activate the DC and induce cell death. Do not attempt to block FcγRII and FcγRIII with anti-CD16/32 antibodies. Blocking antibodies ligate FcγR, induce strong phosphorylation of mitogen-activated protein (MAP) kinases, and prime the DC.
   1. Remove the excess unbound beads by adding 9 mL of isolation buffer and centrifuge cells at 400 rcf for 5 - 10 min at 4^oC.
8. Aspirate the supernatant and re-suspend cells in 1 mL isolation buffer. Apply the cells on a prewashed magnetic column. Wash the column twice with 3 mL of isolation buffer.
   1. Remove the column from the magnet. Pipette 6 mL of isolation buffer onto the column and flush out the magnetically labeled cells into a sterile collection tube by pushing the plunger. Centrifuge cells at 400 rcf for 5 - 10 min at 4 ^oC.
   2. Re-suspend cells at 100 μL per 1 x 10⁷ cells and stain with the following fluorophore-conjugated antibodies: Linage negative (TCRb, Siglec F, B220, CD19, FceRI and Ly6G), MHCII, Ly-6C.
9. Sort cells by gating the small cells, using side scatter (SSC) and forward scatter (FSC), and culture in a complete medium supplemented with 5 ng/mL GM-CSF—Incubate cells for 1 hour at 37 ^oC to allow macrophages to adhere to the plate. Then, transfer the loose and non-adherent cells to a new culture dish.
   NOTE: Mouse MoDC are defined as CD11b⁺/CD11c⁺/MHCII^hi/Ly6C^lo/int. Expression of Ly6C may vary dramatically between tumor models, and in some models (e.g., LMP), MoDCs completely lack Ly6C expression. One 100 mm³ B16F10 tumor typically contains 5 x 10⁴ of DC, resulting in approximately 4 - 5 x 10⁶ total cells. Of these cells, 10 - 15% are immune cells and 8 - 10% are MoDC. Increasing the DC numbers can be achieved by injecting mice with tumors at multiple sites. Sort only small SSC/FCS cells, as other myeloid cells can express markers such as CD11c and Ly6C.        
   OPTIONAL: Sort the tumor-infiltrating monocyte as small SSC/FSC cells that express Ly6C^hi and are negative for MHCII. Afterward, culture monocytes in vitro with 50 ng/mL GM-CSF to obtain DC.

2. Preparation of Tumor-IgG Immune Complexes

1. Culture tumor cells in a 75 cm² culture flask to 70% confluence in complete DMEM media.
   1. Add 2 mL of 0.25% trypsin/EDTA to detach cells from the culture flask and monitor cell morphology under a light microscope to avoid over-trypsinization.
   2. Add 8 mL of complete culture media (per 2 mL of trypsin) to inhibit trypsin digestion, and centrifuge at 4 ^oC, 400 rcf for 5-10 min to pellet the cells.
      NOTE: Check tumor cells for Mycoplasma using a commercial PCR kit. Test for Gram-negative bacteria and fungal endotoxins using Limulus Amebocyte Lysate (LAL) assay). Serum must be filtered through 0.22 μM and tested for the 9 Code of Federal Regulations viruses. Additionally, test the culture media and serums by dropping 100 - 200 μL onto LB agar or broth and culture for 2 days at 37 ^oC.
   3. Wash cells again from trypsin and serum remains by re-suspending them in 10 mL PBS and centrifuge at 400 rcf for 5 - 10 min. Aspirate supernatant and repeat the wash 2 more times.
2. Fix cells in 1.8% buffered paraformaldehyde for 10 min at room temperature.
   1. Wash the cells by re-suspending them in 10 mL PBS and centrifuge at 4 ^oC 400 rcf for 5 - 10 min.
   2. Aspirate supernatants and repeat wash two more times.
      Optional: For tumor uptake assays, cells can be labeled by incubating them for 5 min at 37 ^oC in PBS containing 1 μM carboxyfluorescein succinimidyl ester (CFSE). CFSE is then quenched with complete media for 10 min in ice. The cells should be washed extensively in PBS containing 2% serum to remove residual dye.
3. Re-suspend cells in fluorescence-activated cell sorting (FACS) buffer (PBS supplemented with 2% FCS + 5 mM EDTA) and 0.5 μg/mL of anti-CD16/32 to block potential non-specific protein-protein interactions. Plate in a U-shape 96 wells/plate at a concentration of 1 x 10⁵ cells per 100 μL.
   1. Add different dilutions of tumor-binding antibodies ranging from 5 μg - 5 ng/1 x 10⁵ cells. Incubate the plate on ice for 15-20 min.
      NOTE: IgG antibodies were isolated from the serum of naïve 20-24 weeks old female mice on protein A columns, as described.
4. Wash cells by adding 150 μL of PBS and centrifuging the plate at 4 ^oC 400 rcf for 5 - 10 min.
   1. Discard the supernatants and repeat the wash twice. Re-suspend cells in 100 μL FACS buffer containing fluorophore-conjugated secondary antibody. Incubate plate on ice for 20 min.
   2. Wash cells by adding 200 μL of FACS buffer and centrifuging the plate at 400 rcf for 5 - 10 min. Discard the supernatants and repeat the wash.
   3. Analyze tumor binding by flow cytometry and determine the minimal concentration required to coat the cells.      
      NOTE: Antibodies that do not increase mean fluorescence intensity (MFI) of stained tumor cells by at least fivefold over isotype control should not be used in subsequent functional assays.

3. Activation of MoDC with Tumor-IgG IC

1. Coat tumor cells with minimal IgG concentration, as described in sections 2.1 through 2.4.3
   1. One day before activating MoDC with tumor IC, replace MoDC culture media containing GM-CSF. To do so, gently aspirate the media and wash the cells once with pre-warmed complete culture media.
      NOTE: Isolated mature tumor-associated MoDC should be cultured for at least 2 - 3 h (or even overnight) after sorting in complete media without GM-CSF, and before activating them with IC.
   2. For tumor uptake analyses, add the CFSE-labeled tumor IC to MoDC at a ratio of 1:5 (IC: MoDC) and incubate overnight for 12 - 16 h in 1 mL of complete media per 1 x 10⁶ DC.
   3. For FACS analyses of MoDC activation experiments, add tumor-IC at a 1:1 (IC: MoDC) ratio and incubate overnight for 12 - 16 h.
      NOTE: Including a positive control well is highly recommended, in which MoDC are stimulated with 1 μg/mL of LPS or other TLR agonists.
   4. Following overnight activation, aspirate the supernatants and wash cells gently three times with isolation buffer or 10 mM EDTA HBSS.
   5. To detach DC from the plate, incubate cells for 2 - 3 min in 1 mL HBSS containing 10 mM EDTA and detach cells by vigorous pipetting.
   6. Centrifuge cells at 400 rcf and re-suspend 1 x 10⁶ DC in 90 μL PBS supplemented with 2% FCS, 5 mM EDTA (FACS buffer) and 0.5 μg of blocking antibodies. Incubate on ice for 5 - 10 min.
   7. Add 10 μL of staining antibodies mixture to the cells and incubate on ice for 15 min.
   8. Add 2 mL FACS buffer to cells and centrifuge at 4 ^oC 400 rcf for 5 - 10 min.
2. Re-suspend cells in 200 μL FACS buffer.
   1. Add 0.5 - 1 μg/mL of DAPI 1 - 2 min before running the samples to exclude dead cells from analyses. Do not over-incubate DAPI, as MoDC will take it up within 10 - 15 min.