After reaching 80–90% confluence, the OREC were passaged into 6-well flat-bottom culture plates containing DMEM/F12 medium, without FBS or antibiotics, and were incubated at 37 °C with 5% CO2 for a 24-h starvation treatment. Following this treatment, the OREC were then randomly divided into six groups: five mannan-stimulated groups and one control group. The stimulated groups were exposed to dose titration (10, 50, 100, 200, and 400 μg/mL) of mannan (Sigma, Munich, Germany), while the control group was cultured with DMEM/F12 medium instead of mannan at 37 °C, 5% CO2 for 8 h. Then, the total RNA of each sample was extracted, the cell culture supernatant was collected, and the expression levels of SBD-1 mRNA and protein were detected by qPCR and ELISA, respectively, to determine the optimal concentration of mannan-induced SBD-1 expression.
Mannan
Manufactured by Merck Group
Sourced in United States, Germany, China, Japan, Sao Tome and Principe, Canada
Mannan is a type of carbohydrate polymer found in the cell walls of certain fungi and plants. It serves as a structural component and can be isolated for use in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Laminarin, by Merck Group (7 mentions)
Zymosan, by Merck Group (5 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Laminarin, by InvivoGen (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Depleted zymosan, by InvivoGen (2 mentions)
Dectin 1 mab, by R&D Systems (2 mentions)
Random control sirna, by Santa Cruz Biotechnology (2 mentions)
Epha2 sirna, by Santa Cruz Biotechnology (2 mentions)
Ephb2 sirna, by Santa Cruz Biotechnology (2 mentions)
Ultrapure lipopolysaccharide lps, by InvivoGen (2 mentions)
Cd14 microbead, by Miltenyi Biotec (2 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
Recombinant mouse mr, by R&D Systems (2 mentions)
Immulon 4hbx flat bottom microtiter plates, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Merck Group (2 mentions)
Facsdiva, by BD (2 mentions)
Facsaria, by BD (2 mentions)
79 protocols using mannan
1
Mannan Stimulation of SBD-1 Expression
2
Monoclonal Antibody Cocktail Arthritis Induction
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Cocktails of the monoclonal antibodies including Cab3, anti-CII antibody cocktail (positive control), and isotype antibody cocktail (negative control) were prepared by mixing equal amounts of each sterile filtered antibody solution to achieve a final concentration of 2 or 4 mg. Mice were injected intravenously (i.v.) with the antibody cocktail (Cab3) composed of M2139, L10D9, and 15A11. For the induction of lpsCAIA (Cab-induced and LPS-enhanced arthritis), the mice received (50 μg/mouse) lipopolysaccharide from Escherichia coli O55: B5 (Sigma-Aldrich) intraperitoneally (i.p.) on day 5. For the induction of mCAIA (Cab-induced mannan-enhanced arthritis), the mice received (5 mg/mouse) mannan (Sigma-Aldrich) from Saccharomyces cerevisiae i.p. on days 5 and 60. Signs of arthritis in the paws were followed up macroscopically with blind scoring of each red and swollen joint following a previously described protocol [35 ]. Briefly, macroscopic (clinical) arthritis was defined based on the two following criteria, swelling and redness, and was scored as follows: 1 point was for each inflamed toe or knuckle, whereas 1–5 points were given to an inflamed wrist or ankle according to the severity of disease, resulting in a score of 0 to 15 for each paw and 0 to 60 points for each mouse. Importantly, paws which were still swollen but without erythema were not scored or defined as arthritis.
3
Epithelial Cell Culture and Inhibitor Treatments
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The strains used in the experiments (listed in Supplementary Table 2 ) were grown
as described previously 9 (link). The
OKF6/TERT-2 oral epithelial cell line (provided by Jim Rheinwald,
Dana-Farber/Harvard Cancer Center, Boston, MA) 29 (link) was grown as described 9 (link),29 (link). OKF6/TERT-2 cells have been authenticated by
RNA-Seq3 (link), and have been
tested for mycoplasma contamination. To determine the effects of the various
inhibitors, the host cells were treated for 1 h prior to stimulation or
infection with 2.5 μM dasatinib 30 (link), 400 μM EphA2 antagonist, a
2,5-dimethylpyrrolyl benzoic acid derivative 15 (link),31 (link), 50 μM Stat3 inhibitor S31-201 32 (link), 1μM gefitinib 16 , 3 μg/ml dectin-1 mAb
(R&D Systems) 33 . In other
experiments, the cells were stimulated with 50 μg/ml depleted zymosan
(InvivoGen), 50 μg/ml mannan (Sigma), or 50 μg/ml laminarin
(InvivoGen). To deplete the OKF6/TERT-2 cells of EphA2, they were transfected
with random control siRNA (Santa Cruz Biotechnology; sc-37007), EphA2 siRNA
(Santa Cruz Biotechnology; sc-29304), or EphB2 siRNA (Santa Cruz Biotechnology;
sc-39949) with Lipofectamine 2000 (Invitrogen) following the
manufacturer’s instructions as described previously16 .
as described previously 9 (link). The
OKF6/TERT-2 oral epithelial cell line (provided by Jim Rheinwald,
Dana-Farber/Harvard Cancer Center, Boston, MA) 29 (link) was grown as described 9 (link),29 (link). OKF6/TERT-2 cells have been authenticated by
RNA-Seq3 (link), and have been
tested for mycoplasma contamination. To determine the effects of the various
inhibitors, the host cells were treated for 1 h prior to stimulation or
infection with 2.5 μM dasatinib 30 (link), 400 μM EphA2 antagonist, a
2,5-dimethylpyrrolyl benzoic acid derivative 15 (link),31 (link), 50 μM Stat3 inhibitor S31-201 32 (link), 1μM gefitinib 16 , 3 μg/ml dectin-1 mAb
(R&D Systems) 33 . In other
experiments, the cells were stimulated with 50 μg/ml depleted zymosan
(InvivoGen), 50 μg/ml mannan (Sigma), or 50 μg/ml laminarin
(InvivoGen). To deplete the OKF6/TERT-2 cells of EphA2, they were transfected
with random control siRNA (Santa Cruz Biotechnology; sc-37007), EphA2 siRNA
(Santa Cruz Biotechnology; sc-29304), or EphB2 siRNA (Santa Cruz Biotechnology;
sc-39949) with Lipofectamine 2000 (Invitrogen) following the
manufacturer’s instructions as described previously16 .
4
Lectin Pathway Activation Prothrombin Cleavage
5
Epithelial Cell Culture and Inhibitor Treatments
6
DC-SIGN-Fc Binding ELISA Assay
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High-binding flat-bottom 96-well ELISA plates were coated overnight with 50 mL of 50 mg/mL ligand, i.e. pili sample B, dissolved in 0.2 M coating buffer at room temperature. Mannan (1 mg/mL, Sigma-Aldrich®) was coated as a positive control, whilst plain coating buffer served as negative control. After two washes with TSM (20 mM TrisHCl, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2) the wells were blocked with 1% BSA (Sigma-Aldrich®) for 30 minutes at 37°C. DC-SIGN-Fc fragments were 15 minutes preincubated with blocking agents in TSM: the D1 DC-SIGN specific antibody (20 mg/mL) and calcium chelator EGTA (10 mM). After washing, the plate was incubated with 50 μL of DC-SIGN-Fc (with block) for 2 hours at room temperature on a shaking platform. The wells were washed with TSM with 0.1% Tween 20 (TSMT), prior to the addition of goat anti-human Fc-PO (Sigma-Aldrich®) dissolved in TSMT (30 minutes). After extensive washing with TSMT, the ELISA could be developed using a 10 mL substrate solution of 0.1 M NaAc, TMB and H2O2. 15 minutes later this reaction was stopped by the addition of 0.8 M H2SO4. Experimental read-out was performed at OD450.
7
Candida Phagocytosis Assay with Dectin-1
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To block CR3, adherent RAW-Dectin1 cells were incubated with 10 μg blocking antibody to CD11b (monoclonal M1/70) or rat IgG2B isotype control (MAB0061; R and D systems) for 30 min at room temperature. After warming to 37°C, cells were incubated with Candida-BFP hyphae as described above. Following phagocytosis, monolayers were rinsed and fixed in 3% PFA as mentioned above. After permeabilization and blocking, described above, blocking antibody was detected using a Alexa488-conjugated secondary antibody against rat IgG (1:10,000; Jackson ImmunoResearch Labs), and viewed by confocal microscopy.
For sugar blocking experiments, adherent RAW-Dectin1 cells were pretreated 30 min with 100 μg mL−1 mannan (Sigma Aldrich) or laminarin (Sigma Aldrich), followed by infection with C.albicans in the continued presence of each sugar. In the case of laminarin, Candida-BFP hyphae were allowed to adhere to RAW-Dectin1 cells 10 min, followed by the addition of 100 μg mL−1 laminarin for the remainder of the 1 hr infection.
For sugar blocking experiments, adherent RAW-Dectin1 cells were pretreated 30 min with 100 μg mL−1 mannan (Sigma Aldrich) or laminarin (Sigma Aldrich), followed by infection with C.albicans in the continued presence of each sugar. In the case of laminarin, Candida-BFP hyphae were allowed to adhere to RAW-Dectin1 cells 10 min, followed by the addition of 100 μg mL−1 laminarin for the remainder of the 1 hr infection.
8
Evaluating GAGs Inhibition of Mannan-MBL Interaction
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To evaluate whether GAGs could inhibit the mannan—MBL interaction, Maxisorp immunoassay plates were incubated overnight with 100 μg/ml mannan (Sigma, Zwijndrecht, The Netherlands) diluted in 0,1 M NaCO3, pH 9,6. After washing three times, plates were blocked with 1% BSA in PBS for 1 h. Thereafter, pooled serum diluted 1:50 in GVB++ buffer, described by Roos et al. (39 (link)), were pre-incubated with a concentration range of GAGs, for 15 min at room temperature. GVB++ buffer is Veronal-buffered saline (1,8 mM Na-5,5-diethylbarbital, 0,2 mM 5,5-diethylbarbituric acid, 145 mM NaCl) containing 0,5 mM MgCl2, 2 mM CaCl2, 0,05% Tween-20 and 0,1% gelatin, pH 7.5. The pre-incubated mixture was then incubated for 60 min on the mannan coated plates at 37°C. In the dose dependent binding of MBL to mannan, no GAGs were added. Bound MBL was detected with a DIG-labeled mouse anti-human MBL antibody 1:1,000 (mAb 3e7 from Hycult Biotech, Uden, the Netherlands) and a Sheep anti-DIG HRP labeled conjugate 1:8,000 (Roche Diagnostics, Mannheim, Germany). The assay was developed using tetramethylbenzidine (TMB) (Sigma, Zwijndrecht, The Netherlands) and the reaction was stopped with 1 M H2SO4. Absorbance was measured at 450 nm in a microplate reader. Data was expressed as % inhibition compared to non-inhibited control. Data was presented as representative experiment.
9
Whole Blood Cytokine Assay with Immune Stimuli
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Heparinized venous blood was processed for culture within 6 hours after venipuncture. Whole blood was diluted 2 times in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 100 U/mL penicillin (Astellas Pharma BV, Leiden, the Netherlands), 10 µg/mL streptomycin (Sigma-Aldrich, Saint Louis, MO, USA), 1 mM pyruvate (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich) and stimulated with 50 µg/mL polyinosinic-polycytidylic acid high molecular weight (poly(I:C); InvivoGen, San Diego, CA, USA), 50 ng/mL Pam2CGDPKHPKSF (FSL-1; InvivoGen), 100 ng/mL Pam3CSK4 (Pam3; EMC Microcollections GmbH, Tübingen, Germany), 100 ng/mL ultrapure lipopolysaccharide (LPS; InvivoGen), 10 µg/mL γ-D-Glu-mDAP (iE-DAP; InvivoGen), 100 µg/mL mannan (Sigma-Aldrich), 100 µg/mL curdlan (Wako Chemicals GmbH, Neuss, Germany) or 5 ng/mL 1-(palmitoyl)-2-(5-keto-6-octene-dioyl)phosphatidylcholine (KOdiA-PC; Cayman Chemicals, Ann Arbor, MI, USA), alone or in combination (Table 1 ). Medium was used as a negative control and 2 µg/mL phytohemagglutinin (PHA; Remel, Dartford, UK), a mitogen, as a positive control. 100 µL of ligand(s) in medium was added to wells containing 100 µL of diluted blood in 96-well round-bottom plates (Nunc; Roskilde, Denmark) and incubated in the presence of 5% CO2 at 37°C for 24 hours. Supernatants were harvested and stored at −80°C.
10
Combination Adenovirus Vaccine for Cancer Immunotherapy
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The recombinant adenovirus was coupled with mannan (Sigma) as previously reported56 (link). In brief, adenoviruses were mixed with oxidizing mannan (OM) at a proportion of 1 × 108 PFU:14 mg/ml and incubated overnight at room temperature (RT) for AdTERT-m (AdTERT-mannan) or AdVEGFR2-m (AdVEGFR2-mannan) generation without any further purification. We observed that co-immunization of mice with dendritic cells (DCs) transfected with mRNAs that encode TERT and VEGFR-2 (mixed at a 1:1 ratio) exhibited a synergistic antitumour effect, as reported previously20 (link). We thus used recombinant adenovirus AdTERT-m and AdVEGFR2-m mixed at the ratio of 1:1 (1 × 108 PFU: 1 × 108 PFU) as our combination vaccine Ad(VEGFR2:TERT)-m (Ad(VEGFR2:TERT)-mannan). Additionally, we used phosphate-buffered saline (PBS), Ad-null virus (without the genes of interest) modified with or without OM (Adv-m and Adv, respectively), AdTERT-m alone and AdVEGFR2-m alone as controls.
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