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Tissue digestion c tube

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The Tissue digestion C-tube is a laboratory equipment designed for the mechanical and enzymatic dissociation of tissue samples. It facilitates the disaggregation of solid tissues into a single-cell suspension, which is a crucial step in various cell biology and tissue engineering applications.

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Lab products found in correlation

Gentlemacs dissociator, by Miltenyi Biotec (5 mentions) Ghost dye, by Cytek Biosciences (2 mentions) Collagenase type 3, by Worthington (2 mentions) Albumin rich buffer, by Miltenyi Biotec (2 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions) Collagenase type 1 and 3, by Worthington (2 mentions) Collagenase type 3, by Merck Group (1 mentions) Human cd8 microbeads, by Miltenyi Biotec (1 mentions) 70 μm cell strainer, by BD (1 mentions)

6 protocols using tissue digestion c tube

1

Dissociation of Solid Tumour Samples

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Tumours were cut into small fragments (around 1 mm3) and incubated for 20 minutes with Collagenase Type III (Worthington Biochemical), in RPMI-1640 medium containing 2% FBS (10 ml per gram of tumour tissue) at 37°C.
The tumour pieces were transferred to a tissue digestion C-tube (Miltenyi) and further dissociated enzymatically and mechanically on a gentleMACS Dissociator (Miltenyi). Briefly, the impTumour_03 program was run on the dissociator, followed by a 10-minute incubation at 37°C. Next, the m_Lung_02 program was run to maximize extraction of pericytes and endothelial cells, followed by a 10-minute incubation at 37°C. After a final run of the m_Lung_02 program, the digestion reaction was stopped with albumin-rich buffer (RPMI-1640 medium containing 0.5% bovine serum albumin (BSA)) (Miltenyi). A single cell suspension was obtained by filtering through a 70-μm cell strainer.
Single cell suspensions were centrifuged for 5 minutes at 1,400 g, resuspended in RBC lysis buffer (eBioscience), incubated on ice for 2 minutes, and washed with PBS containing 1.5% FBS (or without FBS if the downstream flow cytometry staining panel included a Ghost Dye (Tonbo)).
Tian L., Goldstein A., Wang H., Lo H.C., Kim I.S., Welte T., Sheng K., Dobrolecki L.E., Zhang X., Putluri N., Phung T.L., Mani S.A., Stossi F., Sreekumar A., Mancini M.A., Decker W.K., Zong C., Lewis M.T, & Zhang X.H. (2017). Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming. Nature, 544(7649), 250-254.
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2

Purification and Characterization of CD8+ T Cells from PDX Tumors

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PDX tumors were cut into ~1 mm3 fragments and incubated for 20 min with collagenase type III (Sigma), in RPMI-1640 medium containing 2% FBS (5 ml/g tumor tissue) at 37 °C. The tumor pieces were transferred to a tissue digestion C-tube (Miltenyi Biotec) and further dissociated enzymatically and mechanically on a gentleMACS Dissociator (Miltenyi Biotec) to generate a single-cell suspension. Afterwards, CD8+ T cells were purified with human CD8 Microbeads (Miltenyi Biotec) or Fluorescence Activating Cell Sorter. Purified cells were subjected to flow cytometry, qRT-PCR, immunoblots, or NF-κB activity assay.
Zhou C., Li W., Liang Z., Wu X., Cheng S., Peng J., Zeng K., Li W., Lan P., Yang X., Xiong L., Zeng Z., Zheng X., Huang L., Fan W., Liu Z., Xing Y., Kang L, & Liu H. (2024). Mutant KRAS-activated circATXN7 fosters tumor immunoescape by sensitizing tumor-specific T cells to activation-induced cell death. Nature Communications, 15, 499.
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3

Dissociation of Solid Tumour Samples

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Tumours were cut into small fragments (around 1 mm3) and incubated for 20 minutes with Collagenase Type III (Worthington Biochemical), in RPMI-1640 medium containing 2% FBS (10 ml per gram of tumour tissue) at 37°C.
The tumour pieces were transferred to a tissue digestion C-tube (Miltenyi) and further dissociated enzymatically and mechanically on a gentleMACS Dissociator (Miltenyi). Briefly, the impTumour_03 program was run on the dissociator, followed by a 10-minute incubation at 37°C. Next, the m_Lung_02 program was run to maximize extraction of pericytes and endothelial cells, followed by a 10-minute incubation at 37°C. After a final run of the m_Lung_02 program, the digestion reaction was stopped with albumin-rich buffer (RPMI-1640 medium containing 0.5% bovine serum albumin (BSA)) (Miltenyi). A single cell suspension was obtained by filtering through a 70-μm cell strainer.
Single cell suspensions were centrifuged for 5 minutes at 1,400 g, resuspended in RBC lysis buffer (eBioscience), incubated on ice for 2 minutes, and washed with PBS containing 1.5% FBS (or without FBS if the downstream flow cytometry staining panel included a Ghost Dye (Tonbo)).
Tian L., Goldstein A., Wang H., Lo H.C., Kim I.S., Welte T., Sheng K., Dobrolecki L.E., Zhang X., Putluri N., Phung T.L., Mani S.A., Stossi F., Sreekumar A., Mancini M.A., Decker W.K., Zong C., Lewis M.T, & Zhang X.H. (2017). Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming. Nature, 544(7649), 250-254.
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4

Primary Cancer Cell Isolation and T Cell Purification

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Tumors were cut into small fragments (approximately 1 mm3) and incubated for 30 min with collagenase type I and III (Worthington Biochemical), in RPMI 1640 medium containing 2% FBS (5 mL/g tumor tissue) at 37 °C. The tumor pieces were transferred to a tissue digestion C-tube (Miltenyi Biotec) and further dissociated enzymatically and mechanically on a gentleMACS Dissociator (Miltenyi Biotec) to obtain a single-cell suspension. Primary cancer cells were purified with EpCAM+ microbeads (Cat. No. 130-061-101, Miltenyi Biotec)24 (link). CD3+ T cells were isolated from peripheral blood with EasySep™ Human T Cell Isolation Kit (Cat. No. 17951, Stemcell) according to the manufacturer’s instructions. The isolated cancer cells were discarded after ten passages.
Lei X., Lin H., Wang J., Ou Z., Ruan Y., Sadagopan A., Chen W., Xie S., Chen B., Li Q., Wang J., Lin H., Zhu X., Yuan X., Tian T., Lv X., Fu S., Zhu X., Zhou J., Pan G., Xia X., Tannous B.A., Ferrone S., Fan S, & Li J. (2022). Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression. Nature Communications, 13, 3882.
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5

Mechanical and Enzymatic Tumor Dissociation

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Tumors were cut into small fragments (around 1 mm3) and transferred to a tissue digestion C‐tube (Miltenyi Biotec) and dissociated enzymatically and mechanically using a tumor dissociation kit (mouse; Miltenyi Biotec) on a gentleMACS. Dissociator (Miltenyi Biotec). Briefly, the “m‐impTumor‐02” program was run on the dissociator, followed by a 40‐min incubation at 37 °C.After a final run of the “m‐impTumor‐03,” a single‐cell suspension was obtained by filtering through a 70‐μm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Single‐cell suspensions were centrifuge for 5 min at 300 g and washed with PBS.
Comunanza V., Gigliotti C., Lamba S., Doronzo G., Vallariello E., Martin V., Isella C., Medico E., Bardelli A., Sangiolo D., Di Nicolantonio F, & Bussolino F. (2023). Dual VEGFA/BRAF targeting boosts PD‐1 blockade in melanoma through GM‐CSF‐mediated infiltration of M1 macrophages. Molecular Oncology, 17(8), 1474-1491.
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6

Isolating Primary Cancer Cells and T Cells

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Tumors were cut into small fragments (approximately 1 mm 3 ) and incubated for 30 minutes with collagenase type I and III (Worthington Biochemical), in RPMI 1640 medium containing 2% FBS (5 ml/g tumor tissue) at 37 ℃. The tumor pieces were transferred to a tissue digestion C-tube (Miltenyi Biotec) and further dissociated enzymatically and mechanically on a gentleMACS Dissociator (Miltenyi Biotec) to obtain a single-cell suspension. Primary cancer cells were puri ed with EpCAM + microbeads (Cat. No. 130-061-101, Miltenyi Biotec) [18] . CD3 + T cells were isolated from peripheral blood with EasySep™ Human T Cell Isolation Kit (Cat. No. 17951, Stemcell) according to the manufacturer's instructions. The isolated cancer cells were discarded after ten passages.
Li J., Lei X., Ruan Y., Lin H., Chen W., Xie S., Li Q., Wang J., Lin H., Zhu X., Yuan X., Ou Z., Tian T., Lv X., Fu S., Zhu X., Zhou J., Pan G., Xia X., Tannous B.A., Ferrone S., & Song F. (2020). Inhibition of Mitochondrial Fission Reverses the Immunoescape of Solid Tumors via IRE1α-XBP-1s-TPP2 axis.
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