2.1. Blood Specimen Collection and Preparation
Peripheral venous blood samples were obtained from healthy adult donors with IRB approval following informed consent and collected in CPT tubes (BD, Franklin Lakes, NJ) with heparin. PBMC were isolated following the manufacture's protocol. Briefly, CPT tubes were centrifuged at 1500 × g for 30 minutes at room temperature. Mononuclear cells were harvested and washed twice with 1% BSA (Celliance, Kankakee, IL) / HBSS (Cellgro, Herndon, VA) by centrifugation at 300 × g for 10 minutes at 4°C. After resuspension in 1% BSA/HBSS, PBMC were adjusted to a final concentration of 20 × 106 cells/ml and kept on ice until stained.
In some experiments, cryopreserved normal control PBMC obtained from one subject with IRB approval were used. In brief, vials containing frozen cells were quickly thawed at 37°C for 65-70 seconds, and cell suspension was transferred to a 15 ml polypropylene at room temperature and diluted by slowly adding 1% BSA / HBSS at room temperature while gently swirling the tube to mix cells thoroughly, then washed twice with 1% BSA / HBSS by centrifugation at 300 × g for 10 minutes at 20°C.
2.2. Antibodies used for flow cytometric analysis
The following monoclonal antibodies were used in this study: CD1c-PE (clone AD5-8F7, Miltenyi Biotec, Auburn, CA), CD3-PE-Cy5.5 (clone S4.1, Caltag/Invitrogen, Carlsbad, CA), CD4-APC-Alexa-fluor 750 (clone RPA-T4, eBioscience, San Diego, CA), CD11c-PE-Cy7 (clone 3.9, Biolegend, San Diego, CA), CD14-Alexa-fluor 700 (clone M5E2, BD Biosciences, San Jose, CA), CD16-PerCp-Cy5.5 (clone 3G8, BD Biosciences, San Jose, CA), CD19-PerCp-Cy5.5 (clone SJ25C1, BD Biosciences, San Jose, CA), CD34-PerCp-Cy5.5 (clone 8G12, BD Biosciences, San Jose, CA), CD40-PE-Cy5 (clone 5C3, BD Biosciences, San Jose, CA), CD86-Pacific Blue (clone IT2.2, Biolegend, San Diego, CA), CD141-biotin (clone AD5-14H12, Miltenyi Biotec, Auburn, CA), CD303-APC (clone AC144, Miltenyi Biotec, Auburn, CA), HLA-DR-PE-Texas Red (clone TU36, Caltag/Invitrogen, Carlsbad, CA). Streptavadin-Pacific Orange (Molecular Probes/Invitrogen, Carlsbad, CA) was used as secondary staining reagent for CD141-biotin. 7-Amino-Actinomycin D (7-AAD) (BD Biosciences, San Jose, CA) was included in the antibody cocktails as a vital dye to exclude dead cells. The following isotype controls were also used: mIgG2a-PE (clone eBM2a, eBioscience, San Diego, CA), mIgG2b-PE-Texas Red (Caltag/Invitrogen, Carlsbad, CA), mIgG1-PE-Cy5 (clone P3, eBioscience, San Diego, CA), mIgG1-PE-Cy7 (clone MOPC-21, Biolegend, San Diego, CA), mIgG2b-Pacific Blue (clone MPC-11, Biolegend, San Diego, CA), mIgG1-Biotin (Caltag/Invitrogen, Carlsbad, CA), mIgG1-APC (clone P3, eBioscience, San Diego, CA), mIgG2a-Alexa-fluor 700 (clone eBM2a, eBioscience, San Diego, CA), and mIgG1-APC-Alexa-fluor 750 (clone P3, eBioscience, San Diego, CA). The antibody cocktails were made by mixing antigen-specific antibodies at optimal concentrations determined by prior titration. Isotype control antibodies were individually substituted for the specific antibody-fluorochrome in the cocktail at a similar antibody concentration. The configuration of lasers and optical filters and the compatible PMT channels for fluorochromes used in the instrument for this study is based on the “6-Blue 2 Violet 0-UV 3-Red Configuration” of the “Common Special Order Configurations” in the BD LSR II User's Guide, with the following minor modifications: we used a 735 LP longpass dichroic mirror for PE-Cy7, a 665 LP longpass dichroic mirror and a 670/14 bandpass filter for PE-Cy5, the Violet PMT A for Pacific Orange, a 440/40 bandpass filter for Pacific Blue, a 735 LP longpass dichroic mirror for APC-Alexa fluor 750, a 685 LP longpass dichroic mirror and a 720/40 longpass filter for Alexa fluor 700. The BD LSR II User's Guide can be obtained from the BD website.
2.3. FITC-dextran uptake assay
The FITC-dextran uptake assay was setup by incubating cells with FITC-dextran in duplicate plates at 4°C and 37°C, respectively. Briefly, 50 μl of PBMC (1 × 106 cells) in 1% BSA/HBSS were added to triplicate wells on each of the two 96-well V-bottom plates (BD, Franklin Lakes, NJ) before adding 4 μl of FITC-dextran (molecular weight = 40,000; Molecular Probes/Invitrogen, Carlsbad, CA) at 12.5 mg/ml for a final concentration of FITC-dextran of 1 mg/ml. The FITC-dextran solution was vortexed for 30 seconds and sonicated for an additional 30 seconds immediately before use. One plate was incubated at 37°C and the second was incubated at 4°C (to determine baseline FITCdextran uptake level) for 30 minutes. Both plates were gently tapped every 5 to 10 minutes to ensure adequate mixing. Following FITC-dextran incubation, 200 μl of 1% BSA/HBSS was added into each well and the plates were spun at 400 × g at 4°C for 6 minutes, decanted supernatant, washed one more time with 250 μl of 1% BSA/HBSS, and followed by cell surface marker staining (see below).
2.4. Cell surface marker staining for flow cytometry analysis
The staining for cell surface markers in this study was done by a “micromethod” that significantly reduces the amount of antibodies and staining volume compared to the standard staining protocol that usually stains one million cells in 100 μl. Briefly, 1 × 106 PBMC per well in the v-bottom plates (BD, Franklin Lakes, NJ) were resuspended with 50 μl of 5% normal mouse serum (Sigma-Aldrich, St. Louis, MO) diluted in 1% BSA/HBSS and incubated at room temperature for 10 minutes to pre-block non-specific staining. After pre-blocking, cells were centrifuged at 400 × g for 6 minutes at 4°C, supernatant was decanted, plate was gently blotted on paper towels, and cell pellets were resuspended with 15 μl of antibody-fluorochrome cocktails containing 20% of Fc receptor blocker (Miltenyi Biotec, Auburn, CA) by thorough but gentle pipetting. Cells and cocktail were incubated on ice for 20 minutes in the dark. Afterwards, cells were washed twice with 200 μl of 1% BSA/HBSS and centrifuged at 400 × g for 6 minutes at 4°C. Cells were then stained with 10 μl of Streptavadin-Pacific Orange at 1 mg/ml, and incubated on ice for 20 minutes in the dark. Following the SA-Pacific orange staining, cells were washed twice as above and resuspended in 200 μl of fixation buffer (2% formaldehyde in DPBS). The staining plate containing fixed cells was loaded onto an 11-color LSR II flow cytometer (BD Biosciences, San Jose, CA) through the High Throughput Sampler (HTS) for data acquisition. The unstained and single stained compensation controls were setup by staining antibody-capturing compensation beads (Simply Cellular anti-mouse compensation standard, Bangs Laboratories, Fishers, IN) with staining buffer alone or the optimal dilution of antibody-fluorochrome conjugate. In average, we acquired minimum 1.5 × 106 total events from triplicate wells for each sample, and about 0.8 × 106 to 1 × 106 leukocytes (based on FSC-SSC gating) minimum were collected, whereas 5000 total events were acquired for compensation beads controls. All flow cytometric data acquired on LSR II were analyzed using FlowJo software (Treestar, Ashland, OR). The raw data files from each of the triplicate samples were concatenated to one single fcs file for analysis. Note: staining was performed using 1 million cells in triplicate wells rather than 3 million cells in one single well following the standard operating procedure established in our laboratory.
2.5. MDC1 and PDC isolation by FACS and in vitro stimulation
To isolate pure MDC1 and PDC subsets for in vitro stimulation, PBMC were stained with an antibody cocktail of CD1c-PE, CD3-PE-Cy5.5, CD4-APC-Alexa Fluor 750, CD11c-PE-Cy7, CD14-PE-Texas Red (clone TUK4, Caltag/Invitrogen, Carlsbad, CA), CD16-PerCP-Cy5.5, CD19-PerCP-Cy5.5, CD34-PerCP-Cy5.5, CD304-APC (clone AD5-17F6, Miltenyi Biotec, Auburn, CA) plus 7-AAD and sorted by FACS on a 7-color FACSAria cell sorter (BD Biosciences, San Jose, CA). Five to six thousand sorted MDC1 (Lin−CD4+CD11c+CD1c+) and PDC (Lin−CD4+CD11c−CD304+) were resuspended in 100 μl of tissue culture medium (RPMI-1640 (Cellgro, Herndon, VA) with 8% of FCS (Sigma-Aldrich, St. Louis, MO) and antibiotic/antimycotic solution) per well in a 96-well v-bottom plate in the presence of 100 ng/ml of LPS from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) or 20 μg/ml of CpG oligodeoxynucleotides 2006 (Coley Pharmaceutical Group, Wellesley, MA) and incubated at 37°C for 18 hours, followed by staining with 7-AAD, CD40-PE-Cy5, and CD86-Pacific Blue. Cells were then fixed and analyzed on a LSR II flow cytometer.
2.6. Visualization of FITC-dextran uptake by imaging flow cytometry (ImageStream)
PBDC were enriched by magnetic beads using the MACS Human Blood Dendritic Cell Isolation Kit II (Miltenyi Biotec, Auburn, CA). The isolated PBDC (a mixture of all three PBDC subsets with purity greater than 93%) were then used to perform the FITC-dextran uptake assay, followed by staining with CD1c-PE or CD303-PE. Cells were analyzed by ImageStream imaging flow cytometry with IDEAS software (Amnis, Seattle, WA). Nuclear dye DRAQ-5 was added to samples 3 minutes before loading samples into the flow cytometer to exclude dead cells.