Fluorescence activated cell sorting (FACS) analysis was performed on isolated ME of the small bowel 3 and 24 h after intestinal manipulation and in untreated control animals as well as in lethally irradiated shielded and non-shielded CX3CR1GFP/+xLysMcre+;Rosa26YFP chimera, respectively. Isolation of ME was achieved by sliding small bowel segments onto a glass rod, removing the outer muscularis circumferentially with moist cotton applicators and cutting the ME into fine pieces. ME was digested with a 0.1% collagenase type II (Worthington Biochemical, Lakewood, NJ, USA) enzyme mixture, diluted in HBSS, containing 0.1 mg/ml DNase I (La Roche, Germany), 2.4 mg/ml Dispase II (La Roche, Germany), 1 mg/ml BSA (Applichem), and 0.7 mg/ml trypsin inhibitor (Applichem) for 40 min in a 37°C shaking water bath. Afterwards single cell suspension was obtained using a 70 µm filter mesh. Cells were stained for 30 min at 4°C with the appropriate antibodies. For antibodies used in this study see Table 1
Trypsin inhibitor
Manufactured by AppliChem
Sourced in Germany
Trypsin inhibitor is a lab equipment product that functions to inhibit the activity of the enzyme trypsin. Trypsin is a serine protease that is commonly used in various biological and biochemical applications. The trypsin inhibitor helps to regulate and control the activity of trypsin in these applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 2, by Worthington (4 mentions)
Dnase 1, by Roche (4 mentions)
Dispase 2, by Roche (4 mentions)
Bovine serum albumin (bsa), by AppliChem (4 mentions)
Facscanto 3, by BD (2 mentions)
Attune nxt, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Leupeptin, by AppliChem (1 mentions)
Pepstatin, by AppliChem (1 mentions)
Gapdh g8795, by Merck Group (1 mentions)
Infrared fluorescent dye conjugated secondary antibodies, by LI COR (1 mentions)
Aprotinin, by AppliChem (1 mentions)
Roti block, by Carl Roth (1 mentions)
Facscanto 1, by BD (1 mentions)
5 protocols using trypsin inhibitor
1
FACS Analysis of Intestinal Muscularis Externa
2
Isolation and Dissociation of Smooth Muscle
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Isolation of ME was achieved by sliding small bowel segments onto a glass rod and removing the outer muscularis circumferentially with moist cotton applicators. ME was then cut into fine pieces and digested with a 0.1% collagenase type II (Worthington Biochemical, Lakewood, NJ, USA) enzyme mixture, diluted in HBSS, containing 0.1 mg/ml DNase I (La Roche, Germany), 2.4 mg/ml Dispase II (La Roche, Germany), 1 mg/ml BSA (Applichem), and 0.7 mg/ml trypsin inhibitor (Applichem) for 40 min in a 37°C shaking water bath. Afterwards single cell suspension was obtained using a 70 µm filter mesh.
3
FACS Analysis of Intestinal Muscularis Externa
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Fluorescence activated cell sorting (FACS) analysis was performed on isolated ME of the small bowel 24 h after intestinal manipulation treated with ambroxol or vehicle CX3CR1‐GFP+/− animals, respectively. Isolation of ME was achieved by sliding small bowel segments onto a glass rod, removing the outer muscularis circumferentially with moist cotton applicators and cutting the ME into fine pieces. ME was digested with a 0.1% collagenase type II (Worthington Biochemical, Lakewood, NJ, USA) enzyme mixture, diluted in HBSS, containing 0.1 mg/ml DNase I (La Roche, Germany), 2.4 mg/ml Dispase II (La Roche, Germany), 1 mg/ml BSA (Applichem), and 0.7 mg/ml trypsin inhibitor (Applichem) for 40 min in a 37°C shaking water bath. Afterward, single cell suspension was obtained using a 70 µm filter mesh. Cells were stained for 30 min at 4°C with the appropriate antibodies. For antibodies used in this study see Appendix Table S4 . Flow cytometry analyses were performed on FACSCanto III (BD Biosciences), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
4
Protein Extraction and Western Blot Analysis
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Cells were lysed in buffer containing 10 mmol/l Tris/HCl, pH 8.0, 150 mmol/l NaCl, 10 mmol/l, 5 mmol/l EDTA, 0.5 % Triton X-100, 60 mmol/l N-octylglucoside, supplemented with 40 mg/l PMSF and protease inhibitor mix (antipain, aprotinin, leupeptin, chymostatin, pepstatin, trypsin inhibitor; 2 mg/ml each; all from AppliChem). After determination of protein concentration by Bradford assay, equal amounts of proteins were boiled in Laemmli buffer and separated by SDS-PAGE gel. After transfer of proteins onto nitrocellulose membranes and blocking with Roti-Block (Carl Roth), the following primary antibodies were used: anti-flotillin-1, flotillin-2 both from BD Biosciences (610821, 610384) and Santa Cruz (sc-25506, sc-25507). Caveolin-1 and caveolin-2 from Abnova (MAB2408) and Cell Signaling (8522). The antibody for VCAM-1(sc-8304), ICAM-1(sc-8439) and p65(sc-109) were from Santa Cruz, GAPDH (G8795) from Sigma-Aldrich. IRF-3 (4302) and pIRF-3 (Ser 385) (4947) were from Cell Signaling. PERK (Thr 981) (sc-32577) and eIF2α (Ser52) (sc-101670) from Santa Cruz. Western blot analysis was performed with an infrared-based laser scanning detection system (Odyssey, Licor). The infrared fluorescent-dye-conjugated secondary antibodies were purchased from Licor.
5
Isolation and FACS Analysis of ME
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FACS analysis was performed on isolated ME samples of the small bowel 24 h after IM in GFAPCrexIL1R1fl/fl animals. Isolation of ME was achieved by sliding small bowel segments onto a glass rod, removing the outer muscularis circumferentially with moist cotton applicators and cutting the ME into fine pieces. ME was digested with a 0.1% collagenase type II (Worthington Biochemical, Lakewood, NJ, USA) enzyme mixture, diluted in PBS, containing 0.1 mg/ml DNase I (La Roche, Germany), 2.4 mg/ml Dispase II (La Roche, Germany), 1 mg/ml BSA (Applichem), and 0.7 mg/ml trypsin inhibitor (Applichem) for 40 min in a 37 °C shaking water bath. Afterwards single-cell suspension was obtained using a 70 µm filter mesh and cells were stained for 30 min at 4 °C with the appropriate antibodies. For antibodies used in this study see Table S4 . Flow cytometry analyses were performed on FACSCantoI(BD Biosciences) using FACSDiva software and data were analyzed with the latest FlowJo software (Tree Star, Ashland, OR, USA).
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