Cls 4

Manufactured by Worthington

Sourced in United States

The CLS-4 is a laboratory centrifuge designed for general-purpose applications. It features a compact design and can accommodate various rotor configurations to suit different sample sizes and types. The CLS-4 provides reliable performance and consistent results for a variety of laboratory procedures.

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Collagenase IV (CLS-4,

6 protocols using cls 4

Isolation of Liver Cells from Mice

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Livers were dissected out taking care to minimize bleeding, and were rinsed with DPBS to wash off excess blood. For wild-type samples, small pieces of liver were dissected out from the left and median lobe (lobe encasing the gallbladder). For knockout samples, liver pieces containing hemangiomas were dissected out. In some cases hemangioma tissue was pooled from two mice to obtain enough material. Next, liver tissue was minced and resuspended in 5–10 ml digestion buffer consisting of ice-cold DMEM with 5 mg/ml (or 800 U/ml) collagenase (trypsin-free Collagenase, CLS-4, Worthington Biochemical Corporation, Lakewood, NY, USA) and 20 mM HEPES. Digestion was performed at 4 °C for 1–1.5 h in 5-ml round bottom polypropylene tubes with overhead rotation. The digest was then diluted 2-fold in FACS buffer, EDTA was added to a final concentration of 2 mM, and the cell solution was strained through a 70-μm strainer. Next, cells were pelleted at 250 xg for 8 min at 4 °C, and resuspended in ice-cold red blood cell lysis buffer (from eBioscience, San Diego, CA, USA). Red blood cell lysis was performed for 3 min on ice. After washing, liver cells were resuspended in FACS buffer and stained as described below.

Bader H.L, & Hsu T. (2016). Inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) in granulocytes contributes to development of liver hemangiomas in a mouse model. BMC Cancer, 16, 797.

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Isolation and Purification of ILC2s

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Lungs were aseptically removed, minced and incubated in 1.6 mg/ml of collagenase (CLS4, Worthington Biochemicals, Lakewood, NJ) at 37°C for 90 minutes and pressed through a 70µM filter mesh. ILC2s were identified as lineage negative (CD3ε, CD11c, CD11b, B220, Ter-119, Gr-1, FcεR1α, γδTCR), CD45⁺, CD127⁺, and ST2⁺ and purified by FACS ARIA flow cytometer (BD Biosciences, Mountain View CA) with a purity of greater than 93%.
Isolation of human ILC2s was performed as previously described (11 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from blood of four donors and then stained with lineage markers (CD3, CD14, CD16, CD19, CD20, CD56, CD235a, CD1a, CD123) and CRTH2, CD161, CD127 and CD45. Thereafter CD45⁺ Lineage⁻ CRTH2⁺ CD127⁺ CD161⁺ population was purified by FACS ARIA flow cytometer with a purity of more than 95%.

Rigas D., Lewis G., Aron J.L., Wang B., Banie H., Sankaranarayanan I., Galle-Treger L., Maazi H., Lo R., Freeman G.J., Sharpe A.H., Soroosh P, & Akbari O. (2016). ILC2 Suppression by Regulatory T Cells Attenuates Airway Hyperreactivity and Requires ICOS:ICOS-Ligand Interaction. The Journal of allergy and clinical immunology, 139(5), 1468-1477.e2.

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Isolation and Purification of Thyroid Cells

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Thyroid tissues were minced for 5 min in Hank’s Balanced Salt Solution (HBSS) and digested with collagenase type 4 (400 unit/mL) (CLS-4, Worthington, Lakewood, NJ, USA) dissolved in RPMI1640 media at 37 °C for 60 min. The cells were centrifuged at 3,100 ×g for 5 min at 4 °C, and the pellets were resuspended in RPMI medium containing 10% fetal bovine serum (FBS). After mechanical dissociation by pipetting, the cell suspension was filtered through a 70-μM cell strainer and centrifuged at 3,100 × g for 5 min at 4 °C. The pellets were incubated in red blood cell lysis buffer (11814389001; Roche Diagnostics, Basel, Switzerland) for 10 min on ice. After centrifugation, the cell pellets were resuspended in FBS-free RPMI medium and filtered through a 40-μM cell strainer^{64 (link)}.

Lee S.E., Park S., Yi S., Choi N.R., Lim M.A., Chang J.W., Won H.R., Kim J.R., Ko H.M., Chung E.J., Park Y.J., Cho S.W., Yu H.W., Choi J.Y., Yeo M.K., Yi B., Yi K., Lim J., Koh J.Y., Lee M.J., Heo J.Y., Yoon S.J., Kwon S.W., Park J.L., Chu I.S., Kim J.M., Kim S.Y., Shan Y., Liu L., Hong S.A., Choi D.W., Park J.O., Ju Y.S., Shong M., Kim S.K., Koo B.S, & Kang Y.E. (2024). Unraveling the role of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer by multi-omics analyses. Nature Communications, 15, 1163.

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Isolation of Rabbit Atrial Myocytes

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Atrial myocytes were freshly isolated from adult New Zealand White rabbits (2.8–3.1 kg) of either gender. Hearts were excised, immediately mounted on a Langendorff column, and perfused for 5 min with Tyrode solutions containing (in mM): 130 NaCl, 5 KCl, 1.8 CaCl₂, 4 KH₂PO₄, 3 MgCl₂, 5 HEPES, 15 taurine, 5 creatine, 10 glucose, pH 7.25, then for 5 min with a Ca²⁺-free solution containing (in mM): 130 NaCl, 5 KCl, 0.5 K₂EGTA, 4 KH₂PO4, 3 MgCl₂, 5 HEPES, 15 taurine, 5 creatine, 10 glucose, pH 7.25. The heart was then perfused with enzyme solution for ~20 min. Atria were excised, minced, and placed in fresh enzyme solution containing collagenase (0.45 mg/ml; Cls 4, Worthington) and pronase (0.015 mg/ml, Type XIV, Sigma-Aldrich) in Ca²⁺-free Tyrode solution without EGTA. The tissue chunks were bubbled with an O₂/CO₂ (95%:5%) gas mixture and gently shaken for two 15-min cycles in a 37 °C shaker bath. At the end of each cycle the supernatant was collected. Isolated myocytes were washed twice and stored in a modified Kraft–Brühe (KB) solution until use. The modified KB solution contained (in mM): 120 K-glutamate, 10 KCl, 10 KH₂PO₄, 0.5 K₂EGTA, 10 taurine, 1.8 MgSO₄, 10 HEPES, 20 glucose, 10 mannitol, pH 7.2 (adjust with KOH). Rod-shaped quiescent cells with clear striations were studied within 10 h of isolation.

Deng W., Mahajan R., Baumgarten C.M, & Logothetis D.E. (2016). The ICl,swell inhibitor DCPIB blocks Kir channels that possess weak affinity for PIP2. Pflugers Archiv : European journal of physiology, 468(5), 817-824.

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Skin and Lymphoid Organ Single-Cell Isolation

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To prepare skin single-cell suspensions, the skin was cleaned, defatted, and digested in 0.25% trypsin (catalog 15050065, Thermo Fisher Scientific) for 20 minutes at 37°C. Epidermis was separated from the dermis using forceps and scalpel blades. The dermis was finely minced and digested for 45 minutes at 37°C with 2 mg/mL collagenase type IV (catalog CLS-4, Worthington Biochemical Corporation) and 1 μg/mL DNase (catalog DN25, MilliporeSigma) in RPMI 1640 (catalog 61870127, Thermo Fisher Scientific) with 5% FBS (catalog 16000044, Thermo Fisher Scientific) in a shaker. The digested skin was then minced, passed over a 70 μm cell strainer (catalog 229483, CELLTREAT Scientific), and washed before staining. Whole spleen or SDLNs were dissociated and filtered with a 70 μm cell strainer. Splenocytes were depleted of erythrocytes by ACK Lysing Buffer (catalog A1049201, Thermo Fisher Scientific) and washed before staining.

Dai Z., Chen J., Chang Y, & Christiano A.M. (2021). Selective inhibition of JAK3 signaling is sufficient to reverse alopecia areata. JCI Insight, 6(7), e142205.

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Isolation of Organoid Units from Mouse Intestine

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The OU isolation process was adapted from our previously published protocol (Sala et al., 2011 (link); Grant et al., 2015 (link); Hou et al., 2018 (link)). Two-week-old C57BL/6 mice were euthanized in a CO₂ chamber and the small intestine was collected and opened longitudinally. The resected tissue was vigorously washed five times with cold Hank's Balanced Salt Solution (HBSS, 14170-112, Life Technology) containing Antibiotic/Antimycotic (Anti/Anti, 15240062, Life Technology) to remove the intestinal content. The intestines were minced into 1 × 1 mm² pieces, washed twice in cold HBSS/Anti/Anti, sedimented at 164 g (Eppendorf 5810R−1,000 rpm) between washes, and digested with type IV collagenase (800 units/ml, CLS-4, Worthington) and dispase (0.12 mg/ml, 17105-041, Gibco) in HBSS for 20 min at 37°C. The digestion was stopped with 10% fetal bovine serum (FBS, Invitrogen) in high-glucose Dulbecco's modified Eagle's medium (DMEM, 10566-016, Invitrogen), the sediment (pellet) was minced with a pipette and centrifuged at 41 g (Eppendorf 5810R−500 rpm) for 5 min to remove single cells. The resulting pellet containing the OU was resuspended in DMEM supplemented with 10% FBS, non-essential amino acids (NEAA, 11140-076, Life Technology) and Anti/Anti, centrifuged one last time at 105 g (Eppendorf 5810R−800 rpm) and the supernatant was removed.

Levin G., Zuber S.M., Squillaro A.I., Sogayar M.C., Grikscheit T.C, & Carreira A.C. (2020). R-Spondin 1 (RSPO1) Increases Mouse Intestinal Organoid Unit Size and Survival in vitro and Improves Tissue-Engineered Small Intestine Formation in vivo. Frontiers in Bioengineering and Biotechnology, 8, 476.

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