Complete imdm

Peyer’s patches were first removed from the small intestines and thereafter, the intestines were opened longitudinally, cut finely and washed vigorously with ice-cold HBSS. The epithelial layer was then disrupted via incubation for 20 minutes in HBSS with 5% FBS, 2.0 mM EDTA and 1 mM DTT at 37 °C with agitation, followed by 20 minutes incubation in HBSS with 5% FBS and 2.0 mM EDTA with agitation. Following each incubation step, tissues were vortexed briefly and the supernatant was discarded. Following, tissues were washed and then minced and incubated at 37 °C for 1.5 hours in complete IMDM (Invitrogen) with 10% FBS, P/S and collagenase D at 1.5 mg/ml (Roche) with shaking. Digested tissues were homogenised and supernatants were first passed through a 100- and then 40-μm cell strainer and pelleted by centrifugation. Leukocytes were then enriched using a discontinuous Percoll (GE healthcare) gradient. After centrifugation at 2,800 rpm for 20 minutes at room temperature with zero deceleration, cells were collected at the 40/70 interface. Collected cells were washed twice and re-suspended in cold PBS with 2% FBS.

Latex beads with a mean particle size of 3 μm (Sigma) were washed with sterile DPBS (Gibco), resuspended in DPBS, and incubated with 1 μg per 10⁷ beads of CD21/CD35 monoclonal antibody (clone 4E3; eBioscience), IgM rat anti-mouse antibody (clone II/41; BD Biosciences), or a combination of both for 2 h at room temperature under continuous mixing on a rotating wheel. As controls, beads were either uncoupled or coupled to the isotype control antibody rat IgG2a kappa isotype control (clone eBR2a; eBioscience). After incubation, the antibody-coupled beads were washed and resuspended in complete IMDM (Gibco, Invitrogen) before incubation with the B cells at an MOI of 7 for 5 h.

Single cell suspensions were prepared by pressing the draining lymph nodes through 70 µM cell-strainers. Cells were resuspended in complete IMDM (Gibco) supplemented with 10% FCS (Gibco) and penicillin and streptomycin (100 U/ml and 100 µg/ml, Gibco). The cells were cultured at 1×10⁶ cells/ml in 96-well plates coated with α-CD3 (20 µg/ml) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Supernatants were collected after 72 h and cytokines were measured by ELISA. Quantities of IL-4, IL-10, IFN-γ and IL-13 were measured by sandwich ELISA as previously described [66] (link).

Bone marrow cells harvested from hDC-SIGN transgenic mice were thawed using IMDM (Lonza, Basel, Switzerland) enriched with 10% FCS (Corning, New York, NY, USA), 50 U/mL penicillin, 50 µg/mL streptomycin (Thermofisher), 50 µM 2-mercaptoethanol (Gibco, Waltham, MA, USA), and Glutamax (Gibco). After washing, the cells were seeded in Petri dishes (Greiner Bio-One, Alphen aan den Rijn, The Netherlands) at a density of 3–5 × 10⁶ cells per dish in 10 mL of complete IMDM (Gibco) with 60 ng/mL X-63. The medium was refreshed on day 2 and day 5 with 60 ng/mL of X-63. Bone marrow-derived dendritic cells (BMDCs) from 7–8-day cultures were utilized in subsequent experiments. These BMDCs were characterized in flow cytometry as cells expressing CD11c⁺ and high levels of MHC-II.

Single cell suspensions from lymph node cells of N. brasiliensis -infected animals were prepared by pressing the MLN through 70 μm cell-strainers. Cells were resuspended in complete IMDM (Gibco) supplemented with 10% FCS (Gibco) and Penicillin and Streptomycin (100 U/ml and 100 μg/ml, Gibco). The cells were cultured at 1×10⁶ cells/ml in 96-well plates coated with α-CD3 (20 μg/ml) or supplemented with NbAg (20 μg/ml) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Supernatants were collected after 72 h and cytokines were measured by sandwich ELISA as previously described [28 (link)].

Single cell suspensions from MLN cells of S. mansoni-infected animals were prepared by pressing the MLN through 70μm cell-strainers. Cells were resuspended in complete IMDM (Gibco) supplemented with 10% FCS (Gibco) and Penicillin and Streptomycin (100 U/ml and 100 μg/ml, Gibco). The cells were cultured at 1×10⁶ cells/ml in 96-well plates coated with α-CD3 (20 μg/ml) or supplemented with SEA (20 μg/ml) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Supernatants were collected after 72 h and cytokines were measured by sandwich ELISA as previously described [33 (link)].

Single cell suspensions were prepared by pressing the draining lymph nodes through 70 μM cell strainers. Cells were resuspended in complete IMDM (Gibco Walthan, MA, USA) supplemented with 10% FCS (Gibco) and penicillin and streptomycin (100 U/ml and 100 μg/ml, Gibco). The cells were cultured at 2 × 10⁶ cells/ml in 48‐well plates coated with α‐CD3 (20 μg/ml) or soluble egg Ag (SEA, 20 μg/ml) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Supernatants were collected after 72 h and cytokines were measured by ELISA. Quantities of IL‐4, IL‐5, IL‐10, IL‐13, and IFN‐γ were measured by sandwich ELISA as previously described.37