Tumors were harvested and cut into small pieces, incubated in 5 mL tumor digestion medium (RPMI, 2% FBS, 10 U/mL DNAse I (Sigma), 200 U/mL collagenase type IV (Life Technologies)) at 37°C for 30 min while shaking. After digestion, tumor digests were then passed through 70 μm cell strainers (Corning), washed once with RPMI containing 10% FBS and once with PBS. Spleens were mechanically dissociated with syringes in RPMI containing 10% FBS and passed through 70 μm cell strainers (Corning). Samples were washed once with PBS and incubated in 2 ml red blood cell lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA in distilled water; all Sigma) for 5 min, followed by twice PBS wash. Lymph nodes were mashed in RPMI containing 10% FBS, and passed through 70 μm cell strainers (Corning), washed once with PBS. All samples were resuspended in the buffer used in downstream experiments.
70 μm cell strainer
Manufactured by Corning
Sourced in United States, Germany
The 70 μm cell strainer is a laboratory equipment designed for filtering and separating cells. It features a mesh size of 70 micrometers, which allows for the efficient removal of larger debris and aggregates from cell suspensions while retaining the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (18 mentions)
Dnase 1, by Merck Group (15 mentions)
Glutamax, by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Dnase 1, by Roche (10 mentions)
Collagenase a, by Roche (8 mentions)
Collagenase d, by Roche (8 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Trypsin inhibitor, by Roche (6 mentions)
Lsrii flow cytometer, by BD (6 mentions)
Collagenase type 4, by Thermo Fisher Scientific (5 mentions)
Facsaria 2, by BD (5 mentions)
Papain, by Roche (5 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (5 mentions)
Hepes, by Thermo Fisher Scientific (5 mentions)
Collagenase type 2, by Worthington (5 mentions)
Red blood cell lysis buffer, by Merck Group (4 mentions)
Collagenase 4, by Merck Group (4 mentions)
Rbc lysis buffer, by BioLegend (4 mentions)
169 protocols using 70 μm cell strainer
1
Tumor and Immune Cell Isolation
2
Intestinal T Cell Isolation Protocol
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Biopsies were collected in HBSS media containing 2% fetal calf serum (FCS) and 0.2% amphotericin B. The intestinal tissue was transferred to HBSS supplemented with 1 mM DTT (Sigma-Aldrich, St Louis, MO) and placed on a rolling device for 10 minutes at 4°C. After discarding the supernatant, the intestinal tissue was transferred to HBSS supplemented with 2% FCS and 5 mM EDTA and shaken (2×) at 180 rpm for 30 minutes at 37°C. The tissue suspension was passed through a 70-μm cell strainer (Costar, Greiner Bio-One, Germany) and constituted the intraepithelial population. To obtain lamina propria T cells, intestinal biopsies were subsequently incubated for 1 hour at 37°C with 1 mg/mL Collagenase IV (Sigma-Aldrich) in RPMI medium (supplemented with 10% FCS, 100 U/mL penicillin-streptomycin, and 0.2% amphotericin B), then forcefully resuspended through a 19G needle, washed, and filtered with 70-μm cell strainer (Costar). The cell suspensions were used for RNA-sequencing after sorting different T cell subsets.
3
Tissue Dissociation and Cell Isolation
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Lungs, lymph nodes, livers, and skeletal muscles (gastrocnemius) were harvested from mice following the indicated treatments. Organs were washed with PBS and separated into single cells. Liver tissue was minced and filtered through a 70-μm cell strainer (Corning). The other tissues were minced and incubated at 37°C for 25 minutes in serum-free DMEM containing collagenase IV (Worthington) and deoxyribonuclease I (Worthington). DMEM supplemented with 10% FCS was used to stop the enzymatic reaction. The cell suspension was filtered through a 70-μm cell strainer (Corning). The cells were then centrifuged for 5 minutes at 500 × g at 10°C. The pellet was reconstituted in Red Cell Lysis Buffer (Sigma-Aldrich) according to the manufacturer's protocol. The tissue-derived single cells were counted and subjected to further assessments.
4
Lymph Node Biopsy Immunophenotyping
5
Xenograft Tumor Cell Enrichment
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A measure of 1 × 106 LNCaP cells in 50 μL medium was mixed with 50 μL Matrigel (BD biosciences, San Jose, CA, US) and was injected into the subcutaneous area of NOD-SCID mice limbs. Mice were checked daily. When LNCaP xenograft tumors volume reached 1.5 cm3, tumors were harvested, minced into 1 mm3 pieces, and then digested with Accumax (Sigma) for 30 min at 37 °C. Digests were poured through a 70 μm cell strainer (Corning, NY, US). Cells were maintained in RPMI-1640 medium with 8% FBS. To separate PSA−/lo and PSA+ cells, as previously described[26] (link), briefly, cells were infected with PSA promoter (PSAP)-GFP vector (which drives eGFP expression) for 72 h. Next, fluorescence-activated cell sorting (FACS) was performed to purify the top 10% GFP-bright (GFP+) and bottom 2%−6% GFP-negative/GFP-dim (GFP− /lo) cells. Through this separating, most purified GFP+cells were PSA+ cells and GFP− cells were PSA− /lo cells.
6
Dissociation of Hep3B and HuH-7 Tumor Samples
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Single‐cell suspensions from Hep3B or HuH‐7 tumor samples were prepared using the following protocol. Whole tumors were minced and incubated in RPMI medium containing collagenase type IV (cat. #LS004188, 1 mg/mL; Worthington Biochemical) and DNaseI (cat. #11284932001, 20 μg/mL; Millipore Sigma) at 37°C for 60 min, then gently cut with scissors, and physically dissociated with the back of the plunger of a 3 mL syringe. The tissues were passed through a 70‐μm cell strainer (Corning). Red blood cells (RBC) were removed by incubating with RBC lysis buffer (BioLegend). A total of 3.0 × 106 cells was stained, and data for 5.0 × 105 cells were collected for each tumor. The cells were stained with antibodies purchased from BioLegend (anti‐CD31 [clone 390], anti‐CD45 [clone 30‐F11], and anti‐PDPN [clone 8.1.1]) and Cell Signaling Technology (anti‐EGFR [clone D1D4J] and Rabbit IgG isotype control [polyclonal]). Cells were also stained with fixable viability dye, and dead cells were gated out from the analysis. The fluorescence of the cells was then analyzed with the flow cytometer FACSLyric and FlowJo software. Tumor cells were determined as CD45‐/CD31‐/PDPN‐. RFI was defined the same way as in vitro analysis.
7
Isolation of Murine Splenic Cells for Fungal Infection Studies
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Immunocompetent C57BL/6J mice infected with A. fumigatus conidia as above were sacrificed at day 1 and 3 post-infection with posterior spleen excision. The excised spleen was milled into small fragments with a plunger end of a syringe and forced through a 70-μm cell strainer (Corning Inc.) Upon washing the cells with cold and sterile PBS, the resulting cell pellet was resuspended in 2 mL of pre-warmed (at 37°C) ACK lysis buffer (0.15 M NH4Cl, 10 Mm KHCO3 and 0.1 mM EDTA). Subsequently, the cells were centrifuged at 1,600 rpm for 5 min at room temperature. Finally, the cells were counted and adjusted to a final concentration of 5x106 cells/mL of pre-warmed RPMI (GIBCO, Thermo Fisher Scientific) enriched with 10% FBS (GIBCO, Thermo Fisher Scientific) and 200 μL of the cellular suspension were seeded in round bottom 96-well plates (Corning Inc.).
8
Liver Nuclei Isolation for Single-Cell ATAC-Seq
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The liver nuclei were isolated using a modified protocol from the Nuclei Isolation for Single Cell ATAC Sequencing (CG000169) Demonstrated Protocol from 10x Genomics. In brief, 200 mg fresh big lobe piece of mouse liver tissue was minced and transferred into a Dounce homogenizer cylinder containing 1 ml of ice-cold homogenization buffer (10 mM NaCl, 10 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 0.1% Tween-20, 0.1% IGEPAL CA-63, 0.01% Digitonin, 1% BSA) and incubated for 5 min on ice. Next, the tissue was homogenized with 15 strokes of pestle A and 15 strokes of pestle B until a homogeneous nucleus suspension was achieved. The resulting homogenate was filtered through a 70 μm cell strainer (Corning). The tissue material was centrifuged at 500g for 5 min at 4 °C and the supernatant was discarded. The tissue pellet was resuspended in 1 ml wash buffer (20 mM NaCl, 20 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 1% BSA). The wash step was repeated one more time and the resulting final pellet was resuspended in 100 µl diluted nucleus buffer (10x Genomics snATAC-seq kit). A total of 9 μl of sample was mixed with 1 μl of AO/PI stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nucleus yield, morphology and the presence of clumps/debris.
9
LUNAR-COV19 Vaccine Evaluation in Mice
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All C57BL/6 mouse studies were performed in accordance with protocols approved by the IACUC at Singapore Health Services, Singapore (ref no.: 2020/SHS/1554). C57BL/6 mice purchased from inVivos were housed in a BSL-2 animal facility at Duke-NUS Medical School. Groups of 6- to 8-week-old wild-type C57BL/6 female mice were vaccinated i.m. with either LUNAR-COV19 or conventional mRNA controls bearing the same SARS-CoV-2 S gene at doses of 0.2 μg, 2 μg, and 10 μg. For transcriptomic and T cell studies, submandibular bleeds were performed for whole blood at 24 h post-vaccination. Day 7 post-immunization, mice were sacrificed, and inguinal lymph nodes and spleens were harvested for investigation of immune gene expression and T cell responses, respectively. Splenocyte suspensions for measuring T cell responses were obtained by crushing spleen through a 70 μm cell strainer (Corning). Red blood cells were removed by lysis using BD PharmLyse reagent. For antibody studies, another set of vaccinated 6- to 8-week-old mice were bled at baseline and at 10, 20, and 30 days post-vaccination.
10
Fetal Thymic Cell Dissociation
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For single cell transfer experiments, microdissected fetal thymic lobes were resuspended in 0.7mg/ml hyaluronidase, 0.35mg/ml collagenase, 0.05mg/ml deoxyribonuclease in PBS for 15 minutes at 37°C. Gentle pipetting was used to aid dissociation and the cells were then spun down and collected in FACS wash (PBS with 5% fetal calf serum [FCS]). For all other experiments, microdissected fetal thymi were dissociated for 5 minutes in TrypLE Express Enzyme (Life Technologies 12604013) in an Eppendorf Thermomixer (1400 rpm, 37°C) and were then triturated with a P1000 and the 25G syringe ten times each. Cell suspensions at 4°C were washed twice in 2% FCS FACS buffer, resuspended as required and filtered through a 70μm cell strainer (Corning) to remove clumps.
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