Fresh tumor samples were obtained soon after sacrificed of the animals. Tumors were minced and digested with a combination of collagenase I (Sangon,China) and DNase I (Sangon, China). PBMCs were derived from patients with Ficoll (GE Healthcare, USA). The cells were stimulated, stained with membrane markers and applied to MACS column for further analysis.
Collagenase 1
Manufactured by Sangon
Sourced in China
Collagenase I is an enzyme used for the dissociation and isolation of cells from tissues. It catalyzes the hydrolysis of collagen, a major structural component of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Sangon (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ficoll, by GE Healthcare (1 mentions)
Hyaluronidase, by Sangon (1 mentions)
Macs dead cell removal kit, by Miltenyi Biotec (1 mentions)
Collagenase 4, by Sangon (1 mentions)
Collagenase 2, by Sangon (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Draq7, by BD (1 mentions)
Ab209960, by Abcam (1 mentions)
Ab199093, by Abcam (1 mentions)
Flow cytometry, by BD (1 mentions)
Human fc block, by BD (1 mentions)
Pma and ionomycin, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm fixation permeabilization solution kit, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
Pentobarbital sodium, by Merck Group (1 mentions)
6 protocols using collagenase 1
1
Tumor Dissociation and PBMC Isolation
2
Tissue Dissociation and Single-Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue was surgically minced on a laboratory sterile table, and tissue fragments were preserved in MACS tissue storage until processing.
The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm3) on ice, and enzymatically digested with 500 U/ml collagenase I (SangonBiotech), 150 U/ml collagenase II (SangonBiotech), 50 U/ml collagenase IV (SangonBiotech), 0.1 mg/ml hyaluronidase (SangonBiotech), 30 U/ml DNaseI (SangonBiotech), and 5% Fetal Bovine Serum Origin South America (Yeasen) for 60 min at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 300 g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).
The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm3) on ice, and enzymatically digested with 500 U/ml collagenase I (SangonBiotech), 150 U/ml collagenase II (SangonBiotech), 50 U/ml collagenase IV (SangonBiotech), 0.1 mg/ml hyaluronidase (SangonBiotech), 30 U/ml DNaseI (SangonBiotech), and 5% Fetal Bovine Serum Origin South America (Yeasen) for 60 min at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 300 g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).
3
Flow Cytometric Analysis of PD-L1 and PD-1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were digested by pancreatin (C125C1, NCM Biotech, Suzhou, Jiangsu, China,) and incubated with PD‐L1‐Alexa Fluor 647 (1:100; ab209960; Abcam, Cambridge, UK,) and the control were incubated with isotype (1:100; ab199093; Abcam) at 4°C for 30 min. After incubation, the cells were washed twice and suspended in phosphate‐buffered saline (PBS). The cells were analyzed by Flow Cytometry (BD Bioscience).
The mouse lung tissues were digested with collagenase I (A004194, Sangon Biotech, Shanghai, China). Cells were stained with fluorescently labeled antibodies. Then cells were analyzed by Flow Cytometry (BD Bioscience), and the data were analyzed using the FlowJo10 software (BD Bioscienc). The following antibodies were used: anti‐CD45 PE/Cy7‐conjugated (1;100;103113;BioLegend, San Diego, CA, USA), anti‐CD3ε PerCP/Cy5.5‐conjugated (1:100;100327; BioLegend), anti‐PD‐1‐APC‐conjugated (1:100; 135209; BioLegend).
The mouse lung tissues were digested with collagenase I (A004194, Sangon Biotech, Shanghai, China). Cells were stained with fluorescently labeled antibodies. Then cells were analyzed by Flow Cytometry (BD Bioscience), and the data were analyzed using the FlowJo10 software (BD Bioscienc). The following antibodies were used: anti‐CD45 PE/Cy7‐conjugated (1;100;103113;BioLegend, San Diego, CA, USA), anti‐CD3ε PerCP/Cy5.5‐conjugated (1:100;100327; BioLegend), anti‐PD‐1‐APC‐conjugated (1:100; 135209; BioLegend).
4
Single-Cell Tumor Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh tumor samples were obtained immediately after surgical resection, of which adipose or necrotic tissues were removed carefully. To collect single‐cell suspensions, tumors were minced and digested in RPMI‐1640 with collagenase I (Sangon; 250 U ml−1) and DNase I (Sangon; 50 U ml−1) for 3 hours at 37 C according to the instructions. Red blood cell lysis buffer was then used for removing red blood cells. After stimulation with cell stimulation cocktail (PMA and ionomycin, eBioscience) and Golgistop (BD Biosciences) for 4 hours, cells were stained with membrane markers in dark for 45 minutes at 4 C after applying Human Fc block (BD Biosciences). Cells were stained with intracellular markers after permeabilization by the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) or BD Pharmingen Transcription Factor Buffer Sets (BD Biosciences). Detailed information on antibodies for flow cytometry is listed in Table S1 . Cells were collected and analyzed on the FACSCelesta (BD Biosciences) in 1 hour. In addition, part of the fresh tumor tissues was formalin fixed and paraffin embedded for MDK IHC staining to stratify the tumor into high/low intratumoral MDK expression groups.
5
Evaluating Cellular Junctions and Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMSO and Triton X-100 were purchased from Abcone (Shanghai, China). Pentobarbital sodium, TNF-α, and Lucifer yellow dye were obtained from Sigma-Aldrich (St. Louis, MO). Primary ZO-1 antibody (mouse anti-rabbit), donkey anti-mouse IgG Alexa Fluor 488, and donkey anti-goat IgG Alexa Fluor 555 were purchased from Thermo Fisher Scientific (Carlsbad, CA). Santa Cruz (Santa Cruz, CA) provided goat-anti-rabbit Cx43 antibodies; HRP-conjugated donkey anti-goat IgG was from Bio-Rad (Hercules, CA). HUABIO (Hangzhou, China) supplied mouse anti-rabbit β-actin antibody, whereas Bio-Rad supplied HRP-conjugated goat anti-mouse IgG (Hercules, CA). Magnetic protein G beads and protein A/G beads were from Millipore (Billerica, MA) and Bimake (Houston, TA) ,repectively. PVDF membrane and 0.25% Trypsin-EDTAwere obtained from Thermo (Carlsbad, CA); ECL kit was provided by Shanghai Epizyme Biomedical Technology Co., Ltd (Shanghai, China); DAPI-mounting media were provided by Vector Laboratories (Burlingame, CA); BSA powders and collagenase I were acquired from Sangon Biotech (Shanghai, China) while DMEM, DMEM/F12, FBS, and Penicillin-Streptomycin (PS) were obtained from Thermo (Carlsbad, CA).
6
Isolation of Mouse Endometrial Stromal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of ESCs was based on the protocol in a previous report [31] . Five-week-old female C57BL/6 mice were injected subcutaneously with 100 µL of the E2 solution (100 ng/100 µL) for 3 consecutive days to stimulate the proliferation of endometrium and killed on the fourth day. The uterine horns were isolated. Uterine horns were transferred to the 0.25% Trypsin-EDTA (Gibco) for 60 min at 4°C, and then incubated for 30 min at 37°C. After incubation, uteri were transfer into a petri dish containing cold DMEM/F12 medium to inactivate trypsin activity, and then vortex for 20 s to release the epithelial sheets. The uteri were transferred to 1mg/mL collagenase I (Sangon Biotech, Shanghai, China) for 30 min at 37°C while shaking (200 rpm). After inhibiting collagenase activity, the stromal cell suspension were centrifuged at 500 x g for 7 min. Cells were re-suspended in DMEM/F-12 with 10% FBS and 1% P/S and then plated on culture dish maintained at 37°C with 5% CO 2 . The non-adherent cells were removed after 6 h of culture.
The medium was changed every other day and passage was conducted when cells reached con uence (80-100%).
The medium was changed every other day and passage was conducted when cells reached con uence (80-100%).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!