Isolation of cells for experiments presented in Fig. 1,2, 4b, 5a-c and 7, and Extended Data Fig. 2a, 4a, c and 5 was performed as previously described 66 . Briefly, hindlimb muscles were excised, minced and digested in collagenase VIII (2 mg/mL, Invitrogen) and dispase (0.5 mg/mL, Gibco 17105-041) for 30 min at 37°C, then filtered throμgh a 70 μM filter and washed twice before resuspension in staining medium. The remaining experiments were performed as previously described 67 . Briefly, hindlimb muscles were excised, minced and digested for 90 min at 37°C in collagenase II (800 units/mL, Gibco 17101-015) in 10 mL/sample of dissociation buffer (Ham's F10 media supplemented with 10% horse serum and 1% penicillin/streptomycin). Samples were washed with 50 mL of dissociation buffer, triturated, and then digested for 30 min at 37°C in collagenase II (200 units/mL) and dispase (11 units/mL) in 10 mL/sample of dissociation buffer. Cell suspensions were passed through a 20-gaμge needle 10 times, washed, filtered through a 40-μm cell strainer, washed, and stained for analysis or sorting by flow cytometry.
Collagenase 8
Manufactured by Thermo Fisher Scientific
Sourced in China
Collagenase VIII is an enzyme used for the digestion of collagen, a structural protein found in various tissues. It is commonly used in cell isolation and tissue dissociation procedures to facilitate the release of cells from their extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase 2, by Thermo Fisher Scientific (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Solarbio (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Anti cd4 clone gk1, by Thermo Fisher Scientific (1 mentions)
Anti cd25 clone pc61, by Thermo Fisher Scientific (1 mentions)
Foxp3 pe clone nrrf 30, by Thermo Fisher Scientific (1 mentions)
4 protocols using collagenase 8
1
Muscle Cell Isolation for Analysis
2
Muscle Cell Isolation Protocol
3
Intestinal T Cell Profiling in Echinococcosis
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Flow cytometry analyses of T lymphocytes in the lamina propria of the small intestine were performed in samples taken from mice infected with either 2000 E. multilocularis protoscolex or PBS on day 90 post infection, according to the protocol described in a previous study [15 (link)]. Briefly, intestines were harvested from mice, cut open longitudinally, and washed in PBS. The fat tissues and Peyer’s Patches (PPs) were removed. Intestines were then cut into 2 cm pieces and washed on a shaker in PBS containing 1 mM DTT for 10 min at 37 °C. After that, the intestines were incubated twice with shaking in PBS containing 30 mM EDTA at 37 °C for 10 min; the fluid was replaced between cycles. Then, the intestines were further cut into 0.5 cm pieces. The tissues were then digested with shaking in RPMI1640 medium (Gibco, Waltham, MA, USA) containing DNase I (Solarbio, Beijing, China) (150 μg/mL) and collagenase VIII (Gibco, Waltham, MA, USA) (200 U/mL) at 37 °C for 70 min. The digested tissues were homogenized by vigorous shaking and then passed through a 70 μm cell strainer to remove large debris. The flow-through was centrifuged in a Percoll gradient at 800× g for 20 min at room temperature and the mononuclear T lymphocytes were harvested from the 40%/80% interphase. Flow cytometry was performed to analyze regulatory T cells (Treg) according to a previous study [16 (link)].
4
Isolation and Characterization of Lamina Propria Lymphocytes
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The colon tissue selected from 0.5 cm below the cecum to 0.5 cm above the anus was cut longitudinally and then transversely into 0.5 cm pieces after removing adipose tissue, mesenteric connective tissue, and Peyer's patches. After washing with PBS, colon pieces were digested with collagenase solution containing 1 mg/mL collagenase VIII and 1 U/mL DNase I (Gibco, Life Technologies) for 55 min in a 37 °C shaker. The supernatant was filtered with 40 μm cell strainer, and centrifuged at 2000 rpm for 5 min. The cell suspension was centrifuged after washing with plain RPMI 1640. Lamina propria lymphocytes (LPLs) were separated by density gradient centrifugation (cells were resuspended with 40% Percoll solution, and overlaid with an 80% Percoll solution, at 2500 rpm, 25 min). The interface containing the LPLs were aspirated and washed in medium, and subjected to stain with anti-CD4 (clone GK1.5; eBioscience), anti-CD25 (clone PC61; eBioscience) at 4 °C in the dark for 30 min. Cells were fixed and permeabilized with 200 μL fixation-permeabilization buffer overnight at 4 °C in the dark. Subsequently, cells were incubated intracellularly with Foxp3-PE (clone NRRF-30; eBioscience) at 4 °C in the dark for 1 h, and were analyzed by flow cytometry (Cyto Flex S). The data were analyzed by FlowJo software (Tree Star).
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