Bone marrow was isolated by washing the femur shaft with PBS. Cells were passed through a 70 μm nylon filter (BD) and red cell lysis was performed. Perfused livers were passed through a 70 μm nylon filter (BD) and lymphocytes were purified by a Percoll (GE healthcare) gradient centrifugation. Lungs were minced with razor blades and incubated in PBS containing 60 U/mL DNase (AppliChem) and 170 U/mL collagenase II (Gibco) at 37°C for 45 min. Cell aggregates were dispersed by passing the digest through a 70 μm nylon filter (BD).
70 μm nylon filter
Manufactured by BD
Sourced in United States
The 70 μm nylon filter is a laboratory equipment used for filtration purposes. It has a pore size of 70 micrometers and is made of nylon material.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (4 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (4 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Collagenase a, by Roche (3 mentions)
Liberase tl, by Roche (3 mentions)
Ack lysing buffer, by Lonza (2 mentions)
Dnase, by Roche (2 mentions)
Dissociation enzymes, by Miltenyi Biotec (2 mentions)
Rpmi 1640 medium, by Welgene (2 mentions)
Dmem f 12 glutamax media, by Thermo Fisher Scientific (2 mentions)
24 well tissue culture plate, by BD (2 mentions)
Nerve growth factor (ngf), by Merck Group (2 mentions)
Ara c, by Merck Group (2 mentions)
Collagenase d, by Roche (2 mentions)
Trypsin inhibitor, by Roche (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
35 protocols using 70 μm nylon filter
1
Isolation of Tissue-Resident Immune Cells
2
Isolation and Characterization of fMSCs
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fMSCs were dissociated with 0.05% trypsin-EDTA at 37 °C for 5 min. The ovary tissues were dissected, washed in PBS, and then enzymatically digested to single-cell suspensions (0.25% trypsin-EDTA for 15 min). Single-cell suspensions were passed through a 70-μm nylon filter (BD, USA). After washing with PBS, the collected cells were suspended in buffer (0.1% BSA in PBS). To analyze cell surface markers on fMSCs, the dissociated cells were stained with the following PE-conjugated antibodies: anti-CD105, anti-CD29, anti-CD73, anti-CD90, anti-CD34, and anti-CD45, all purchased from Becton Dickinson and Company (USA). The hGCs and cell suspensions from ovarian tissues were stained with anti-KI67 (BD, USA), anti-ROS (Abcam, USA), or its corresponding isotype control at 4 °C for 30 min. The stained cells were analyzed with a flow cytometer (Beckman, USA) using the manufacturer’s directions.
3
Skin Tissue Dissociation and Flow Cytometry
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Single-cell suspensions were prepared by mincing skin tissue with scissors, followed by a 60-min enzymatic digestion with 2 mg/ml collagenase Type II (Worthington), 2 mg/ml collagenase Type IV (Gibco), and 0.5 mg/ml DNase I (Roche) in PBS containing 1% bovine serum albumin (Sigma) at 37 °C under continuous stirring conditions. Digests were quenched by adding DMEM medium (Gibco) containing 10% heat-inactivated FBS and subsequently filtered through a 70-μm nylon filter (BD Biosciences). Cells were washed with DMEM before being stained with 1 μg/ml of anti-CD3, anti-NK1.1, anti-Gr-1, and anti-F4/80 monoclonal antibodies (BioLegend) according to the manufacturer’s instructions. Cells were analyzed with the Guava EasyCyte 8HT two laser, 6-color microcapillary-based benchtop flow cytometer (Millipore).
4
Tumor Single-Cell Isolation and Analysis
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Tumors were first weighed and then minced with scissors in RPMI and filtered through a 70-μm nylon filter (BD Biosciences) in RPMI to generate single-cell suspensions. The suspensions were purified on a Ficoll gradient to eliminate dead cells and treated with red blood cell lysis buffer (ACK Lysing Buffer, Lonza) and further washed and resuspended in FACS buffer (PBS/0.5% albumin) before incubation with antibodies. The antibodies used for staining are listed in Supplemental Table 2 . In specified experiments, cell numbers were calculated per gram of tumor, and the ratio of CD8 to CD11b calculated from the absolute number of cells/gram of tumor. In experiments to detect SA-Spider-gal by flow cytometry, Spider β-Gal kits (Dojindo Laboratories) were used per the manufacturer’s protocol, prior to staining with antibodies. In experiments to study TCRs, tetramers of the following epitopes were used: hGP10025–33 (KVPRNQDWL)–Alexa Fluor 488 and OT-1 SIINFEKL-PE (NIH). Samples were acquired on a Cytek Aurora flow cytometer. Data analyses were performed using FlowJo v10 (FlowJo).
5
Isolation and Characterization of Murine Bone Marrow and Splenocytes
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Mice (5 animals per group per time-point) were sacrificed on days 10, 15 and 30 of the experiment (or as indicated); two femurs and the spleen were collected from each mouse. Whole bone marrow was harvested from the femurs by flushing the marrow cavity with X-Vivo 10 medium (Lonza, Verviers, Belgium). To isolate splenocytes, spleens were pushed through a sterile 70 μm cell strainer (Corning Incorporated, Corning, NY) in PBS. Single-cell suspensions of bone marrow or splenocytes were obtained by passing cells through an 18-gauge needle. The cells were centrifuged at 350 x g for 10 min. Erythrocytes were removed by lysis in hypotonic ammonium chloride (AKC lysing buffer) for 5 min at room temperature. The cells were washed twice in PBS, and after centrifugation, were resuspended in X-Vivo 10 medium supplemented with 1x Antibiotic Antimycotic Solution (Sigma-Aldrich Co., St. Louis, MO) passed over a 70 μm nylon filter (BD Biosciences, San Jose, CA) and counted with a Fuchs-Rosenthal counting chamber. Viability was accessed by trypan blue staining. Solutions of 1 x 107 cells/ml (or as indicated) were used for colony-forming cell assays.
6
Muscle Dissociation and Cell Isolation
7
Isolation and Culture of Dental Pulp Cells
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Dental pulp explants were digested with 3 mg/ml collagenase type I and 4 mg/ml dispase (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h. The cell suspension was centrifuged at 300 g for 10 min, washed and then filtered through a 70 μm nylon filter (BD Biosciences, San Jose, CA, USA). Dental pulp cells were maintained in α-modified Eagle's medium (α-MEM) supplemented with 10 % fetal bovine serum (FBS) (Gibco-Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (Sigma-Aldrich) at 37 °C in a humidified atmosphere with 5 % CO2 for 3 weeks. The medium was renewed every 3 days.
8
Evaluating Bacterial Growth and Dissemination
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To determine bacterial growth and dissemination levels, the aerobic bacteria in blood, peritoneal lavage fluids, and spleens were assessed.
Six hours after CLP or sham surgery, blood was collected in sterile tubes with previously added EDTA for anticoagulation. Whole blood was diluted 10- to 104-fold with sterile PBS, and 20 μL was immediately plated on 5% solid agar medium for bacterial culture. The remaining portion was centrifuged at 3,000 rpm for 8 min, and the plasma was then isolated for further experiments.
Mice peritoneum lavage fluids were obtained in 5 mL of ice-cold PBS. Lavage fluids were collected and filtered through a 70-μm nylon filter (BD Biosciences) to remove debris. Peritoneal lavage fluids from mice that underwent sham surgery were diluted by 10-fold, whereas fluids from mice that underwent CLP surgery were diluted by 102–104-fold with sterile PBS and 20 μL of each were immediately plated on solid medium for bacterial culture. The remaining portions were stored at −80°C for future experiments (21 (link)).
To evaluate the bacterial seeding in organs, spleens were excised and homogenized in 1 mL sterile PBS. After vortexing, 20 μL of the fluids were immediately used for bacterial culture (21 (link)).
All solid media were incubated overnight at 37°C, and colony-forming units (CFUs) were calculated according to the dilution fold.
Six hours after CLP or sham surgery, blood was collected in sterile tubes with previously added EDTA for anticoagulation. Whole blood was diluted 10- to 104-fold with sterile PBS, and 20 μL was immediately plated on 5% solid agar medium for bacterial culture. The remaining portion was centrifuged at 3,000 rpm for 8 min, and the plasma was then isolated for further experiments.
Mice peritoneum lavage fluids were obtained in 5 mL of ice-cold PBS. Lavage fluids were collected and filtered through a 70-μm nylon filter (BD Biosciences) to remove debris. Peritoneal lavage fluids from mice that underwent sham surgery were diluted by 10-fold, whereas fluids from mice that underwent CLP surgery were diluted by 102–104-fold with sterile PBS and 20 μL of each were immediately plated on solid medium for bacterial culture. The remaining portions were stored at −80°C for future experiments (21 (link)).
To evaluate the bacterial seeding in organs, spleens were excised and homogenized in 1 mL sterile PBS. After vortexing, 20 μL of the fluids were immediately used for bacterial culture (21 (link)).
All solid media were incubated overnight at 37°C, and colony-forming units (CFUs) were calculated according to the dilution fold.
9
Isolation of Endothelial and Hematopoietic Cells
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E11.5 embryos were minced using sharp scissors and placed in 500 μl 4 mg/ml collagenase IV (Life Technologies) and 0.2 mg/ml DNaseI (Roche), 10% FBS (Life Technologies) in PBS, and incubated for 15 min at 37°C while shaking at 600 rpm. The tissue was further disrupted by repeated pipetting using a 1‐ml plastic pipette, every 5 min. Enzyme activity was quenched by adding 1 ml of FACS buffer (PBS, 0.5% FBS, 2 mM EDTA), and the solution was passed through a 70‐μm nylon filter (BD biosciences). Cells were collected and resuspended in 100 μl FACS buffer containing Fc receptor antibody (Invitrogen, 14‐0161‐85, clone 93) for 15 min. After FcR blocking, cells were incubated with directly conjugated antibodies FITC‐CD31 (BD Pharmingen, 553372, clone MEC 13.3) and APC‐CD45 (BioLegend, 103112, clone 30‐F11) staining for 30 min at room temperature. Cells were then washed and resuspended in FACS buffer to be read by the BD CytoFLEX flow cytometer (Beckman Coulter) at the Uppsala University BioVis platform.
10
Splenocyte Cytokine Profiling Assay
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The day of sacrifice, spleens were removed and placed in RPMI medium for culture (Breyner et al., 2017 (link)). Briefly, spleens were crushed and filtered through a 70 μm nylon filter (BD, Le Pont-de-Claix, France) and were resuspended in RPMI (Lonza, Levallois-Perret, France) completed with 100 Unit of Streptomycin, Penicillin and 10% Fetal Calf Serum (FCS). Red blood cells were removed with buffered Blood Cell Lysis Buffer (Sigma-Aldrich, France). Plates were pre-coated with anti-CD3 and anti-CD28 antibodies, 4 μg/ml of each antibody (eBioscience) in PBS with 0.5% FCS. Splenocytes cells were adjusted to 2 × 106 cells/ml per well and were cultured in 24-well plates at 37°C in a 5% CO2 and 95% air atmosphere. Supernatants were collected after 48 h of culture and were stored at -80°C until further analysis. Splenocytes culture supernatant levels of IL-4, IL-5 (Mabtech, Stockholm, Sweden), IL-6, IFN-γ (eBioscience), and IL-10 (BioLegend, San Diego, CA, United States) were quantified by ELISA, following manufacturer instructions.
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