Mandibular dLNs of CD11c-YFP mice were excised, and single cells were obtained by mechanically passing the samples through a 40 μm strainer (Thermo Fisher Scientific, Waltham, MA, United States). Single-cell suspensions were then blocked in FACS buffer containing 1% anti-CD16/CD32 Fc block (Bio X Cell) and were stained with a viability marker (LIVE/DEAD Fixable Blue Dead Cell Stain kit, Thermo Fisher Scientific) for 30 min at RT. Next, samples were stained with fluorophore-conjugated antibodies against CD45, CD11c, dendritic cell inhibitory receptor 2 (DCIR2), CD68, CD3, or respective isotype controls (all BioLegend, or BD Biosciences, San Jose, CA, United States) for 45 min at RT to yield fluorescence minus one stainings. After washing, samples underwent flow cytometric acquisition using a BD LSR II flow cytometer (BD Biosciences). Postacquisition data analysis was performed using FlowJo v9 software (FlowJo LLC, Ashland, OR, United States).
40 μm strainer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 40 μm strainer is a laboratory filtration device designed to remove particles or contaminants larger than 40 micrometers from liquid or semi-solid samples. It is commonly used in various research and analytical applications to separate and isolate specific components from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Gene pulser xcell electroporater, by Bio-Rad (2 mentions)
Gene pulser electroporation cuvettes, by Bio-Rad (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Collagenase hyaluronidase, by STEMCELL (2 mentions)
Chromium system, by 10x Genomics (2 mentions)
Nextseq 500 machine, by Illumina (2 mentions)
Nuclei ez lysis buffer, by Merck Group (2 mentions)
Biocoat plates, by Corning (2 mentions)
Irfp670, by Addgene (2 mentions)
Moflo xdp cell sorter, by Beckman Coulter (2 mentions)
Optiprep density gradient, by Merck Group (2 mentions)
Hibernate a without calcium, by Transnetyx (2 mentions)
Papain, by Worthington (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Fungizone, by Thermo Fisher Scientific (2 mentions)
Complete media, by Avantor (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
21 protocols using 40 μm strainer
1
Flow Cytometry Analysis of Dendritic Cells
2
CRISPR-Mediated Genome Editing in LCLs
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LCL cells were electroporated using a Gene Pulser Xcell Electroporater (BioRad) and 0.4 cm gap Gene Pulser electroporation cuvettes (BioRad). Briefly, 1 × 107 LCL cells were resuspended in 250 μL RPMI-160 plus GlutaMax. To this media was added 65 μg of plasmid expressing ABE7.10, GFP, and the corresponding sgRNA targeting the C282Y mutation in the HFE gene. The mixture was added to a pre-chilled 0.4 cm gap electroporation cuvette and the cell/DNA mixture was incubated in the cuvette on ice for 10 min. Cells were pulsed at 250 V and 950 μF for 3 ms. Cells were transferred back on ice for 10 min, then transferred to 15 mL of pre-warmed RPMI-160 supplemented with 20% FBS in a T-75 flask. The next day, an additional 5 mL of media was added to the flask and cells were left to incubate for a total of 5 days. After incubation, cells were isolated by centrifugation, resuspended in 400 μL of media, filtered through a 40 μm strainer (Thermo Fisher Scientific), and sorted for GFP fluorescence using an FACSAria III Flow Cytometer (Becton Dickenson Biosciences). GFP-positive cells were collected in a 1.5-mL tube containing 500 μL of media. After centrifugation, the media was removed and cells were washed twice with 600 μL of 1× PBS (Thermo Fisher Scientific). Genomic DNA was extracted as described above.
3
Vesicle Isolation from Cell Aggregates
4
Generation of Bone Marrow Chimera
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Generation of bone marrow chimera was as described (51 (link)) in C57BL/6J mice. Retroviral constructs were generated by Epoch Life Science custom cloning services. Bone marrow cells were collected, cultured, transduced with ELF4WT and ELF4T187N retrovirus, purified by cell sorting, and injected intravenously into lethally irradiated Elf4–/– or Elf4+/+ recipients. Three months later, blood was collected to confirm NK reconstitution, and mice were euthanized to collect spleens 24 hours poststimulation. Tissue from mice was processed on ice, and cells were isolated by mechanical separation using a 40 μm strainer (Thermo Fisher Scientific). RBCs from the single-cell suspension pellet were lysed by deionized water, followed by quickly recovering isotonic conditions with 4× PBS. Additional details can be found in the Supplemental Methods .
5
CRISPR-Mediated Genome Editing in LCLs
6
Sphere Formation Assay for Cell Cultures
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Sphere formation assays were performed as previously described [76 (link)]. In brief, Cells were detached from culture plates using accutase (Biolegend, San Diego, CA, USA) and passed through a 25 G needle three times and a 40-μm strainer (Thermo Fisher Scientific) to obtain a single cell suspension. A total of 10,000 cells were seeded per well of a six-well plate coated with 2% polyhema (Sigma). After 7 days, spheres with size of ≥ 100 μm were counted.
7
Isolation and Permeabilization of Nuclei
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Nuclei were isolated and permeabilized in accordance with 10x Genomics’ protocol for Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing. Briefly, tissue was minced into smaller fragments and then put into NP40 lysis buffer (Thermo Fisher Scientific, catalog PI28324). Tissue was homogenized with a pellet pestle 15 times by hand, then incubated in the lysis buffer for 5 minutes. The suspension was then passed through a 70 μm strainer followed by a 40 μm strainer (Thermo Fisher Scientific). The suspension was then centrifuged at 500g for 5 minutes at 4°C. The supernatant was removed, and the pellet was washed twice with PBS + 1% BSA and centrifuged at 500g for 5 minutes at 4°C after each wash. The pellet was then incubated in 0.1× lysis buffer and incubated for 2 minutes to permeabilize the sample. The sample was then centrifuged at 500g for 5 minutes at 4°C; supernatant was removed and resuspended in diluted nuclei buffer.
8
Single-cell Nuclei Isolation and Sequencing
9
Immune Cell Isolation from Tumor Samples
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Tumors were established and harvested as previously described and dissociated either mechanically or mechanical/enzymatic digestion (gentleMACSTM Dissociator) before being filtered through a 40μM strainer (Thermo Fisher Scientific, Waltham, MA, USA). Zombie Aqua or UV Fixable Viability dye (Zombie dye, BioLegend, San Diego, CA, USA) was used to discriminate dead cells (Zombie positive). Anti-mouse CD45.2 monoclonal antibody (clone 104, 109830, BioLegend, San Diego, CA, USA, dilution 1:100, host: mouse,) was used to label immune cells. Samples were acquired using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo (Tree Star, version 10.5.2, BD Life Sciences, Ashland, Oregon, USA). FACS before scRNA-seq was performed using a Beckman Coulter MoFlo ASTRIOS BSL-2 cell sorter at the FACS Lab, Department of BioMedical Research (DBMR), University of Bern, Bern, Switzerland.
10
Vesicle Isolation from Cell Aggregates
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D10–14 aggregates were washed with DMEM/F12 and transferred to a low-attachment dish with 1× collagenase/hyaluronidase (Stemcell Technologies). Aggregates were incubated at 37 °C for 45 min and mechanically disrupted every 15 min. After 45 min, aggregates were disrupted with an uncut 1 mL pipet tip and reverse filtered through a 40 μm strainer (Thermo Fisher) into a low-attachment dish using DMEM/ F12. Vesicles were manually collected within 30 min of disruption and transferred into droplets of 100% Matrigel covered in MM or into MM supplemented with lower concentrations of non-polymerized Matrigel.
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