DC2.4 cells were seeded in complete RPMI at 75% confluency in 24-well plates and DC2.4 cells were stimulated with 10 ng/ml recombinant TGF-beta.
Growth protocol
DC2.4 cells were purchased from Millipore Sigma and were cultured in complete RPMI medium containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 ug/ml), sodium pyruvate (1 mM), L-glutamine (2 mM), beta-mercaptoethanol (50 µM), and HEPES (100 mM). All cells were cultured in an incubator at 37°C, 5% CO2, and 95% relative humidity.In seperate experiment, mice were subcutaneously injected with either 1 x 106 live MCA101 tumor cells in the shaved right flank. Thereafter, tumors were cut into small pieces and were digested in 20 ml of RPMI containing collagenase D (400 mg/ml), collagenase VIII (400 mg/ml), and 2% FCS for 1 hour at 37ºC. Subsequently, 70% and 30% percoll density gradients were used to remove debris and dead cells, and single cell suspensions were collected from the gradient interface. Spleens and tumor-draining lymph nodes (dLNs) were cut and dissociated with 10 U/ml collagenase I and 30 U/mL DNase I (Millipore Sigma) for 45 min at 37°C, and single cell suspensions were collected by passing cells through 70-micron cell strainers. Red blood cells from all samples were removed using erythrocyte lysis buffer. Single cells suspensions from spleen and tumor (MCA101) were stained with antibodies directed against CD11c, MHC-II, CD103, CD8, F4/80, CD11b and CD45, and CD103-pos/CD8-pos DC1 cells purified using FACS sorting. DC2.4 cells were collected upon trypsinization after 4 days of 2.5 ng/ml TGF-beta stimulation. Expression of genes involved in glycosylation were quantified by quantitative real-time PCR (qRT-PCR) using the glycosylation RT2 Profiler PCR Array according to the manufacturer’s instructions (PAMM-046Z, Qiagen). Briefly, RNA was isolated using the RNeasy Kit (Qiagen), and 50-100 ng RNA was converted to cDNA using the RT2 First Strand Kit (Qiagen). The cDNAs were subjected to amplification using gene-specific primers and SYBR Green (RT2 SYBR Green Master Mix, Qiagen) in an Ab7100 Real-Time PCR System (Thermo Fisher Scientific).
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using the RNeasy Kit (Qiagen)
Label
SYBR Green
Label protocol
n/a
Hybridization protocol
n/a
Scan protocol
n/a
Description
Tumor DCs
Data processing
Amplification of the gene encoding for beta-actin served as internal control, and relative gene expression levels were calculated using the 2-ΔΔCT method.
