Experiments were performed as described [19 (link)]. In brief, the dorsal skin of individual mice that were anesthetized with isoflurane was shaved, followed by application of 400μL of 10mg/mL FITC dissolved in 1:1 acetone/dibutylphthalate (Sigma-Aldrich). After 24 hours, mouse inguinal and axillary lymph nodes were isolated by digestion at 37°C for 1h with a cocktail of 100U/ml DNase I (fraction IX; Sigma-Aldrich) and 1.6 mg/mL collagenase (CLS4; Worthington Biochemical). In the experiment using Slamf8-Fc fusion, 100μg of Slamf8-Fc fusion protein or human IgG-Fc (Jackson ImmunoResearch Laboratories) was intraperitoneally injected 3 hours before 200μL FITC application. Single-cell suspensions were stained and analyzed by flow cytometry.
Acetone dibutyl phthalate
Manufactured by Merck Group
Sourced in United States
Acetone/dibutyl phthalate is a laboratory reagent used as a solvent. It is a clear, colorless liquid with a characteristic odor. The product is primarily used in chemical analysis and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein isothiocyanate (fitc), by Merck Group (2 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase cls4, by Worthington (1 mentions)
Transwell chamber, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Roche (1 mentions)
Otc compound, by Sakura Finetek (1 mentions)
5 protocols using acetone dibutyl phthalate
1
FITC-induced Contact Hypersensitivity Assay
2
FITC-Induced Chronic Skin Inflammation Model
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BALB/c mice were randomized to the following groups: vehicle cream b.i.d., 1.5% w/w ruxolitinib cream q.d., 1.5% w/w ruxolitinib cream b.i.d. and 0.05% w/w betamethasone cream q.d. and dosed on both ears with 20 mg cream per ear for the duration of the study.
On day 0, mice were sensitized on the shaved abdomen with 100 µl of 0.5% w/v fluorescein isothiocyanate (FITC) in acetone-dibutyl phthalate 1:1 (v:v) solution (Sigma Aldrich, USA). Sensitization was repeated on days 1 and 2. On day 7 mice were challenged with 20 µl of FITC solution on the right ear. Ear re-challenge was repeated once per week for 4 additional weeks to induce chronic skin inflammation. Ear swelling was measured at 24 and 72 h post FITC challenge. At termination, ear biopsies were weighed and RNA was isolated. Right draining (auricular) lymph nodes were excised for immunophenotyping.
On day 0, mice were sensitized on the shaved abdomen with 100 µl of 0.5% w/v fluorescein isothiocyanate (FITC) in acetone-dibutyl phthalate 1:1 (v:v) solution (Sigma Aldrich, USA). Sensitization was repeated on days 1 and 2. On day 7 mice were challenged with 20 µl of FITC solution on the right ear. Ear re-challenge was repeated once per week for 4 additional weeks to induce chronic skin inflammation. Ear swelling was measured at 24 and 72 h post FITC challenge. At termination, ear biopsies were weighed and RNA was isolated. Right draining (auricular) lymph nodes were excised for immunophenotyping.
3
In Vitro Migration Assay for Dendritic Cells
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The in vitro migration assay was performed using 24-well Transwell chambers with 8-μm pores (Corning, NY, USA). The RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Shanghai, China) and 50 ng/ml CCL21 was added into the lower chamber, and BMDCs with a density of 1 × 105 cells/200 μl were seeded into the upper chamber and incubated for 18 h at 37 °C. In selected experiments DCs were pretreated with vehicle or 4 μg/ml of an Akt agonist, SC79 (HY-18749; MedChemExpress, Shanghai, China), for 1 h. After incubation, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of membrane penetrated cells was determined in the randomly selected fields.
For fluorescein isothiocyanate (FITC)-induced DC migration, mice were painted with 200 μl FITC (10 mg/ml) (46950; Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in acetone/dibutyl phthalate (1:1, Sigma-Aldrich Co.) mixture on the shaved abdomen. The number of DCs migrated into the inguinal lymph nodes was analyzed by flow cytometry 24 h later.
For fluorescein isothiocyanate (FITC)-induced DC migration, mice were painted with 200 μl FITC (10 mg/ml) (46950; Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in acetone/dibutyl phthalate (1:1, Sigma-Aldrich Co.) mixture on the shaved abdomen. The number of DCs migrated into the inguinal lymph nodes was analyzed by flow cytometry 24 h later.
4
Contact Hypersensitivity Assay in Mice
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Twenty-five microliters of 0.5% (v/v) DNFB in acetone/olive oil (4:1) solution was applied onto the shaved abdomen of the mice on days 0 and 1 (sensitization phase). On day 5, the right ear was treated with 20 μL 0.2% DNFB (10 μL on each side of the pinna), and the same for the left ear with 20 μL acetone/olive oil (challenge phase). Ear swelling was measured with a digimatic thickness gauge (Mitsutoyo, Kanagawa-ken, Japan) before and after 24 to 48 h DNFB challenge. The CHS response (ear swelling) was calculated by subtracting pre-challenge ear thickness for each animal. After euthanasia at the indicated days, the ears were removed and fixed with 10% formaldehyde in PBS and embedded in paraffin for hematoxylin and eosin staining, or were unfixed and embedded in OTC compound (Sakura Finetek, Torrance, CA), and processed for immunohistochemistry staining. In vivo DC migration was performed on shaved abdomen as described52 (link). Briefly, fluorescein isothiocyanate (FITC, Sigma) 4 mg/mL was dissolved in 1:1 acetone:dibutyl phthalate (Sigma) and 50 µL applied to the abdominal skin. Recovered inguinal and axillary LNs were digested in 2 mg/mL collagenase D (Roche) for 30 min at 37 °C. Cells were then passed through a 100 µm cell strainer, and stained for flow cytometry.
5
FITC Painting and Tape Stripping
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We anesthetized mice for 5 min and painted their ears with 30 μl of 1% FITC (Sigma-Aldrich) in a carrier solution of acetone/dibutyl phthalate (1:1; Sigma-Aldrich and J. T. Barker). After 20 hours, we collected ipsilateral draining cervical LNs and contralateral nondraining cervical LNs and obtained single-cell suspensions. They were stained for CD11c and analyzed by flow cytometry after LIVE/DEAD staining. For ear tape stripping, we stripped an ear of WT or Cacnb3−/− mice 10 times with ordinary adhesive tape. After 20 hours, we collected ipsilateral draining and contralateral nondraining cervical LNs and quantified the number of migrated migDCs by flow cytometry as described above.
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