Collagenase 8, by Merck Group - Product details

1

Isolation of Intestinal and Lymphoid Cells

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For the isolation of cells from the small intestinal and colonic lamina propria, the tissues were cut longitudinally and into sections following the removal of fat. Peyer’s Patches (PPs) were removed from SI samples. The tissues were incubated for two rounds in HBSS with 2 mM EDTA at 37 °C, for 20 min for the SI or 15 min for the colon, in a shaking incubator. The SI was digested in complete RPMI supplemented with 1 mg/ml Collagenase VIII (Sigma) at 37 °C for ~15 min. The colon was digested in complete RPMI containing 0.85 mg/ml Collagenase VIII (Sigma), 1.25 mg/ml Collagenase D (Roche, Switzerland), 1 mg/ml Dispase (Gibco) and 30 μg/ml DNAse (Roche) at 37 °C for ~40 min. After the digest, cells were passed through a 100 and 40 μm cell strainers.
LNs were cut into smaller pieces and digested for 20 min at 37 °C in RPMI supplemented with 0.075 mg/ml DNAseI (Roche) and 0.75 mg/ml Collagenase-Dispase (Roche). After the digest, cells were passed through a 40 μm cell strainer.
Lymph cells were passed through a 40 μm cell strainer.

Kästele V., Mayer J., Lee E.S., Papazian N., Cole J.J., Cerovic V., Belz G., Tomura M., Eberl G., Goodyear C., Maciewicz R.A., Wall D., Cupedo T., Withers D.R, & Milling S. (2021). Intestinal-derived ILCs migrating in lymph increase IFNγ production in response to Salmonella Typhimurium infection. Mucosal Immunology, 14(3), 717-727.

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2

Isolation of Murine Immune Cells

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SPLs and mesenteric lymph nodes (mLNs) were harvested in HBSS with Ca²⁺ and Mg²⁺ supplemented with 2% fetal calf serum and 10 mM HEPES. For CD4⁺ T cell investigation, SPL and mLN cell suspensions were prepared with frosted slides and filtered through nylon mesh after red blood cell lysis. For analyses of innate immune cell, SPL and mLN suspensions were obtained by collagenase VIII digestion (Sigma-Aldrich, St. Louis, MO) for 30 minutes at 37°C. Colonic lamina propria lymphocyte suspensions were obtained as previously described^{21 (link)}: colons were flushed in phosphate-buffered saline 1× and opened longitudinally. To remove intraepithelial lymphocytes and epithelial cells, intestinal pieces were incubated in HBSS without Ca²⁺/Mg²⁺ supplemented with 10 mM EDTA, 10 mM HEPES, 0.5% fetal calf serum, and 1.5 mM DTET, for 2 × 20 minutes at 37°C. Intestinal pieces were digested in HBSS with Ca²⁺/Mg²⁺, 20% fetal calf serum, 100 U/mL collagenase VIII, and 5 μg/mL DNase (Sigma-Aldrich) for 60 to 90 minutes at 37°C. Lamina propria lymphocytes were purified over a 40% to 100% Percoll gradient (GE Healthcare Bio-Sciences Corp, Piscataway, NJ).

Wurbel M.A., Bras S.L., Ibourk M., Pardo M., McIntire M.G., Coco D., Geha R.S., Fiebiger E, & Snapper S.B. (2014). CCL25/CCR9 Interactions Are Not Essential for Colitis Development but Are Required for Innate Immune Cell Protection from Chronic Experimental Murine Colitis. Inflammatory bowel diseases, 20(7), 1165-1176.

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3

Splenocyte Proliferation Assay

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Spleens were digested with collagenase 8 (Sigma) and DNase-I for 45 min and red blood cells were lysed using ACK buffer (Gibco). Splenocytes were labeled with 12.5µg/ml CFSE (Invitrogen) for 10min at 37°C and immediately underlayed with 3ml FCS to spin out CSFE. Cells were taken up in media (RPMI/10%FCS/L-glutamine/Pen-Strep/HEPES/Sodium pyruvate/ β-mercaptoethanol), counted, and seeded at 200,000 cells per well in round-bottom 96-well plates. Cells were incubated in media with various concentrations of CpG-B, R848, or LPS for 72h. Flow cytometry was used to analyze stimulated cells. Live, singlet cells were pre-gated on CD19⁺ and cell proliferation was determined by the geometric mean fluorescence intensity (gMFI) of CFSE (Extended Data Fig. 10c). For the quantification, a proliferation index was determined by dividing the gMFI CSFE of the unstimulated control by the gMFI CSFE of the stimulated sample (CSFE^Unstim:CFSE^Sample) and plotted along the ligand titration.

Majer O., Liu B., Woo B.J., Kreuk L.S., Van Dis E, & Barton G.M. (2019). Release from Unc93b1 reinforces the compartmentalized activation of select TLRs. Nature, 575(7782), 371-374.

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4

Splenocyte Proliferation Assay

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Spleens were digested with collagenase 8 (Sigma) and DNase-I for 45 min and red blood cells were lysed using ACK buffer (Gibco). Splenocytes were labeled with 12.5µg/ml CFSE (Invitrogen) for 10min at 37°C and immediately underlayed with 3ml FCS to spin out CSFE. Cells were taken up in media (RPMI/10%FCS/L-glutamine/Pen-Strep/HEPES/Sodium pyruvate/ β-mercaptoethanol), counted, and seeded at 200,000 cells per well in round-bottom 96-well plates. Cells were incubated in media with various concentrations of CpG-B, R848, or LPS for 72h. Flow cytometry was used to analyze stimulated cells. Live, singlet cells were pre-gated on CD19⁺ and cell proliferation was determined by the geometric mean fluorescence intensity (gMFI) of CFSE (Extended Data Fig. 10c). For the quantification, a proliferation index was determined by dividing the gMFI CSFE of the unstimulated control by the gMFI CSFE of the stimulated sample (CSFE^Unstim:CFSE^Sample) and plotted along the ligand titration.

Majer O., Liu B., Woo B.J., Kreuk L.S., Van Dis E, & Barton G.M. (2019). Release from Unc93b1 reinforces the compartmentalized activation of select TLRs. Nature, 575(7782), 371-374.

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5

Splenocyte Proliferation Assay with TLR Ligands

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Spleens were digested with collagenase 8 (Sigma) and DNase-I for 45 min and red blood cells were lysed using ACK buffer (Gibco). Splenocytes were labeled with 12.5ug/ml CFSE (Invitrogen) for 10 min at 37°C and immediately underlayed with 3 ml FCS to spin out CSFE. Cells were taken up in media (RPMI/10%FCS/L-glutamine/Pen-Strep/HEPES/Sodium pyruvate/β-mercaptoethanol), counted, and seeded at 200,000 cells per well in round-bottom 96-well plates. Cells were incubated in media with various concentrations of CpG-B, R848, or LPS for 72 h. Flow cytometry was used to analyze stimulated cells. Live, singlet cells were pre-gated on CD19⁺ and cell proliferation was determined by the geometric mean fluorescence intensity (gMFI) of CFSE. For the quantification, a proliferation index was determined by dividing the gMFI CSFE of the unstimulated control by the gMFI CSFE of the stimulated sample (CSFE^Unstim:CFSE^Sample) and plotted along the ligand titration.

Majer O., Liu B., Kreuk L.S., Krogan N, & Barton G.M. (2019). Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity. Nature, 575(7782), 366-370.

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6

Splenocyte Proliferation Assay with TLR Ligands

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Spleens were digested with collagenase 8 (Sigma) and DNase-I for 45 min and red blood cells were lysed using ACK buffer (Gibco). Splenocytes were labeled with 12.5ug/ml CFSE (Invitrogen) for 10 min at 37°C and immediately underlayed with 3 ml FCS to spin out CSFE. Cells were taken up in media (RPMI/10%FCS/L-glutamine/Pen-Strep/HEPES/Sodium pyruvate/β-mercaptoethanol), counted, and seeded at 200,000 cells per well in round-bottom 96-well plates. Cells were incubated in media with various concentrations of CpG-B, R848, or LPS for 72 h. Flow cytometry was used to analyze stimulated cells. Live, singlet cells were pre-gated on CD19⁺ and cell proliferation was determined by the geometric mean fluorescence intensity (gMFI) of CFSE. For the quantification, a proliferation index was determined by dividing the gMFI CSFE of the unstimulated control by the gMFI CSFE of the stimulated sample (CSFE^Unstim:CFSE^Sample) and plotted along the ligand titration.

Majer O., Liu B., Kreuk L.S., Krogan N, & Barton G.M. (2019). Unc93b1 recruits Syntenin-1 to dampen TLR7 signaling and prevent autoimmunity. Nature, 575(7782), 366-370.

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7

Isolation of Tumor-Infiltrating Leukocytes

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Upon excision, tumors were finely minced and incubated in HBSS (Gibco) supplemented with 2% FCS (HBSS-2), Collagenase 8 at 0.05mg/mL (Sigma), 1mM sodium pyruvate (Gibco), 25mM HEPES (Thermo Fisher), and DNaseI at 10mg/mL (Roche) at 37C on a shaker at 80RPM for 30 minutes. The mixture was then thoroughly titrated and passed through a 70um filter and neutralized with HBSS-2 to dilute the collagenase. Tumor-infiltrating leukocytes were subsequently purified via density gradient centrifugation using Percoll (GE Healthcare). Briefly, the cells were resuspended in 35% Percoll and then 70% Percoll was added to the bottom of the suspension by a glass Pasteur pipette. The suspension was spun at 2100 RPM for 20 minutes at room temperature with the brake off. After the spin, the pellet of red blood cells was removed, as well as excess Percoll/buffer, leaving a purified population of tumor-infiltrating leukocytes at the interface. The isolated leukocytes were washed twice with HBSS-2 prior to staining.

Tavazoie M.F., Pollack I., Tanqueco R., Ostendorf B.N., Reis B.S., Gonsalves F.C., Kurth I., Andreu-agullo C., Derbyshire M.L., Posada J., Takeda S., Tafreshian K.N., Rowinsky E., Szarek M., Waltzman R.J., Mcmillan E.A., Zhao C., Mita M., Mita A., Chmielowski B., Postow M.A., Ribas A., Mucida D, & Tavazoie S.F. (2018). LXR/ApoE activation restricts innate immune suppression in cancer. Cell, 172(4), 825-840.e18.

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8

Splenocyte Isolation and Flow Cytometry

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Spleens were minced with scissors and digested for 45 minutes with 50 mg/mL collagenase 8 (Sigma) and 5 ug/mL DNase (Sigma) in RPMI. Tissue was dissociated through a 70 micron filter, treated with ACK Lysis Buffer (Gibco) to eliminate red blood cells, and then resuspended in flow cytometry buffer. For flow cytometry, cells were stained with antibodies to CD11c (Tonbo clone N418) and dead cells were excluded using DAPI. Data were collected on a LSR Fortessa (BD Biosciences) and analyzed using FlowJo software.

Price A.E., Shamardani K., Lugo K.A., Deguine J., Roberts A.W., Lee B.L, & Barton G.M. (2018). A map of Toll-like receptor expression in the intestinal epithelium reveals distinct spatial, cell type-specific, and temporal patterns. Immunity, 49(3), 560-575.e6.

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9

Isolation of CD45- Stromal Cells

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For CD45^- SC isolation, mLNs or peripheral lymph nodes (pLNs) were obtained from the wild-type mice, and the LNs were digested at 37 °C for 30 min with 1 mg/ml collagenase 8 (Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640/10% FCS. The CD45^- cells were isolated using the MACS technique in accordance with the instructions provided by Miltenyi (Bergisch-Gladbach, Germany). The mean purity of the CD45^- cells from the mLN and pLN cells was 97.4% ± 2.0 and 97.5% ± 2.2, respectively, and that of the stromal cell subsets was 88% ± 1.5. The SCs were used for mRNA isolation.

Streich K., Smoczek M., Hegermann J., Dittrich-Breiholz O., Bornemann M., Siebert A., Bleich A, & Buettner M. (2020). Dietary lipids accumulate in macrophages and stromal cells and change the microarchitecture of mesenteric lymph nodes. Journal of Advanced Research, 24, 291-300.

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10

Isolation and Stimulation of Murine Immune Cells

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Spleens and Peyer’s patches (PP) were removed and passed through a cell strainer (90 µm, Falcon) to obtain a single cell suspension. Splenocytes were treated with red blood cell lysing buffer (Sigma-Aldrich), washed and resuspended in complete medium: RPMI 1640 with FCS (10%, Gibco) and penicillin–streptomycin (100 U/ml and 100 µg/ml, respectively, HyClone). Cells from PP were cultured in complete medium but with additional gentamicin (50 µg/ml, Gibco) to prevent bacterial contamination. After isolation, cells from spleen and PP (3–5 × 10⁶ cells/ml) were either left unstimulated or polyclonally stimulated with αCD3 (1 µg/ml, Biolegend) and αCD28 (5 µg/ml, BioLegend) for 24 h. Cell culture supernatants were harvested and stored at −80°C.
Colonic lamina propria (cLP) leukocytes were isolated as previously described (17 (link)). In brief, colon tissue was incubated with EDTA (5 mM) to eliminate epithelial cells, followed by digestion with collagenase 8 (0.5 mg/ml, Sigma Aldrich) and DNase 1 (40 µg/ml, Roche). The released cells were layered on a 30/40/70% Percoll gradient (GE Healthcare).

Petursdottir D.H., Nordlander S., Qazi K.R., Carvalho-Queiroz C., Ahmed Osman O., Hell E., Björkander S., Haileselassie Y., Navis M., Kokkinou E., Lio I.Z., Hennemann J., Brodin B., Huseby D.L., Nilsson C., Hughes D., Udekwu K.I, & Sverremark-Ekström E. (2017). Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice. Frontiers in Immunology, 8, 1699.

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Collagenase 8

Lab products found in correlation

Close spelling variants of this product

192 protocols using collagenase 8

Isolation of Intestinal and Lymphoid Cells

Isolation of Murine Immune Cells

Splenocyte Proliferation Assay

Splenocyte Proliferation Assay

Splenocyte Proliferation Assay with TLR Ligands

Splenocyte Proliferation Assay with TLR Ligands

Isolation of Tumor-Infiltrating Leukocytes

Splenocyte Isolation and Flow Cytometry

Isolation of CD45- Stromal Cells

Isolation and Stimulation of Murine Immune Cells

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