On day 23 after tumor implantation, tumor tissues were resected, chopped into small pieces, and digested in a 37 °C and 5% CO2 incubator for 20 min with fresh RPMI-1640 (Biowest, L0498) containing collagenase type IV (Worthington Biochemical, LS004189, 1 mg/mL), hyaluronidase (Sigma, H6254, 1 mg/mL), and DNase I (Sigma, DN25, 1.5 mg/mL). Digested tissues were minced, filtered through a 70-μm cell strainer, and used to prepare single-cell suspensions according to the manufacturer’s instructions.
H6254
Manufactured by Merck Group
Sourced in United States
H6254 is a laboratory equipment product offered by Merck Group. It serves as a high-performance centrifuge, designed for versatile applications in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mouse tumor dissociation kit, by Miltenyi Biotec (2 mentions)
C2674, by Merck Group (2 mentions)
70 μm cell strainer, by BD (2 mentions)
Rlt buffer, by Qiagen (2 mentions)
Ls004189, by Merck Group (1 mentions)
Rpmi 1640, by Biowest (1 mentions)
C9891, by Merck Group (1 mentions)
Ficoll gradient, by GE Healthcare (1 mentions)
D5025, by Merck Group (1 mentions)
H l alexa fluor 594, by Abcam (1 mentions)
H l alexa fluor 488, by Abcam (1 mentions)
Eclipse ti s microscope, by Nikon (1 mentions)
Ii ii6b3, by Developmental Studies Hybridoma Bank (1 mentions)
5 protocols using h6254
1
Tumor Tissue Dissociation Protocol
2
Tumor Tissue Dissociation for Flow Cytometry
3
Lung Metastasis Immune Cell Analysis
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Vaccinated and control mice with LLC tumors metastasized to lungs were euthanized 35 days after tumor challenge. Lungs were resected and metastasized LLC tumors were dissected and chopped into small pieces before incubation with a mixture of enzymes dissolved in HBSS, including collagenase type IV (400 U/mL; C9891; Sigma-Aldrich; St. Louis, MO, USA), hyaluronidase (0.025 mg/mL; H6254; Sigma-Aldrich) and DNase I (0.01 mg/mL; D5025; Sigma-Aldrich) for 30 minutes at 37 °C with occasional shaking. The resultant cells were washed and passed through a Ficoll gradient (17144002; GE Healthcare; Chicago, IL, USA) to eliminate dead cells. TILs were then analyzed by flow cytometry for the expression of markers for different immune cells. Anti-CD45 antibody was used to selectively exclude CD45− tumor cells from analysis so that only CD45+ immune cells were evaluated. The same number of cells (based on side-scatter and forward-scatter analyses) was acquired in all samples. Respective antibodies specific for the markers were used to quantitate the abundance of different immune cell types. T regulatory cells (Tregs; Foxp3+) were analyzed using the anti-mouse Foxp3 staining kit (00-5523-00; Thermo Fisher).
4
Immunohistochemistry of Collagens I, II, and X
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5 µm thick paraffin-embedded pellets were deparaffinized, rehydrated, and then treated with protease XXV (AP-9006-005, Thermo Scientific) and hyaluronidase (H6254, Sigma-Aldrich). Sections were then incubated with primary antibody: rabbit anti-collagen I (CL50111AP-1, Cedarlane, ON, Canada), mouse anti-collagen II (II-II6B3, Developmental Studies Hybridoma Bank, IA, USA) using a 1:200 dilution and rabbit anti-collagen X (58632, Abcam, UK) using 1:100 dilution at 4 °C overnight, followed by incubation with a goat anti-rabbit IgG (H&L Alexa Fluor 594, Abcam, UK) with a 1:200 dilution for collagen I, X and goat anti-mouse IgG (H&L Alexa Fluor 488, Abcam) with a 1:200 dilution for collagen II. Sections were then stained with DAPI (4′, 6-diamidino-2-phenylindole, Cedarlane) and mounted with Glycerol and PBS (1:1 ratio). Immunofluorescence was visualized by an Eclipse Ti-S microscope (Nikon Canada, Mississauga, Canada).
5
Tumor Tissue Dissociation for Flow Cytometry
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Unless stated otherwise, tumors were excised 21 days after transplantation. For subsequent analysis by flow cytometry, tumors were cut into pieces and digested with either Collagenase Ia (1mg/mL) C2674 Sigma Aldrich and Hyaluronidase V (0.1mg/mL) H6254 Sigma Aldrich for 40min at 37°C or with a mouse tumor dissociation kit (Miltenyi Biotec #130-096-730) using gentle MACS dissociator. Tissue was passed through a 70μm cell strainer (Falcon) and washed with FACS buffer (PBS with 1% FCS) before proceeding with antibody staining. For RNA isolation, homogenization was performed in RLT buffer (QIAGEN) facilitated by a closed tissue grinder system (Fisher brand #02-542-09, 15mL).
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