CT26 or WEHI-164 tumor-bearing mice were vaccinated as described above. The injection of F8-TNF was postponed by 4 days, in order to ensure an adequate tumor size for the analysis of tumor infiltrating lymphocytes. Tumor draining lymph nodes (DLN) and tumors were excised on the day after the first F8-TNF injection. DLN were minced in PBS, treated RBC Lysis Buffer (Biolegend), passed through a 40 μm cell-strainer (EASYstrainer, greiner bio-one) and repeatedly washed. The single cell suspension was used directly for flow cytometric analyses. Tumors were cut into small pieces and digested in RPMI-1640 (Thermo Fisher, with L-glutamine) containing antibiotic-antimycotic solution (Thermo Fisher), 1 mg/mL collagenase II (Thermo Fisher) and 0.1 mg/mL DNase I (Roche) at 37ºC, 5% CO2 for 2 h in an orbital shaker set to 110 rpm. The cell suspension was then passed through a 40 μm cell-strainer (EASYstrainer, greiner bio-one), repeatedly washed and immediately used for flow cytometric analyses.
Easystrainer
Manufactured by Greiner
Sourced in Austria, Germany, France
The EASYstrainer is a lab equipment product designed to assist with filtration tasks. It features a stainless steel mesh screen that can be used to separate solid materials from liquids. The EASYstrainer is a functional tool intended for laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (2 mentions)
Collagenase type 1, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Methocult gf m3434 medium, by STEMCELL (1 mentions)
Murine il 3, by Immunotools (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Immunotools (1 mentions)
Stem cell factor (scf), by Immunotools (1 mentions)
Cx3cr1, by BioLegend (1 mentions)
Cd127, by BioLegend (1 mentions)
Cd49a, by BioLegend (1 mentions)
Cxcr3, by BioLegend (1 mentions)
Il 23r, by BioLegend (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd49b, by BioLegend (1 mentions)
Cd11b, by BioLegend (1 mentions)
Liberase tl, by Roche (1 mentions)
27 protocols using easystrainer
1
Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes
2
Aspergillus fumigatus Conidial Harvesting Protocol
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To obtain fresh conidia, Aspergillus fumigatus was routinely grown in 25-cm2 culture flasks containing 10 ml of 4% potato dextrose agar (PDA) and grown at 37°C for 3 days, unless stated otherwise. Conidia were either inoculated by spreading 20 μl conidial suspension evenly on the medium with a loop or by inoculating with a 2-μl drop of conidial suspension in the middle of the medium. Conidia were harvested by adding 10 ml saline Tween (ST) (0.9% NaCl plus 0.005% Tween 80) to culture flasks that were shaken vigorously. In the case of problems encountered suspending conidia (e.g., with melanin mutants), conidia were collected in ST by scraping with a loop. The ΔchsE, ΔchsEb, and Δpmt4 mutants needed to be grown on PDA supplemented with 6% KCl, since they showed no or little sporulation on PDA. Therefore, the corresponding control ku80 pyrG+ strain was also grown on PDA plus 6% KCl as a control for these strains. Conidial suspensions were filtered through 40- to 100-μm filters (EASYstrainer; Greiner Bio-One).
To obtain isotropically grown conidia or germlings, 108 conidia were inoculated in 25-cm2 culture flasks containing 20 ml liquid minimal medium (MM) plus 1% glucose and incubated for 6 h at 37°C or 16 h at 30°C, respectively, as standing cultures. Cells were harvested by shaking and vortexing, and cultures were filtered through 100-μm filters (EASYstrainer; Greiner Bio-One).
To obtain isotropically grown conidia or germlings, 108 conidia were inoculated in 25-cm2 culture flasks containing 20 ml liquid minimal medium (MM) plus 1% glucose and incubated for 6 h at 37°C or 16 h at 30°C, respectively, as standing cultures. Cells were harvested by shaking and vortexing, and cultures were filtered through 100-μm filters (EASYstrainer; Greiner Bio-One).
3
Generation of Mouse AML Cell Lines
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For generation of mouse AML cell lines, primary tumors (spleen, bone marrow) were mechanically disrupted by mashing through 70 μm EASYstrainer (Greiner). After erythrocyte lysis (5 min at RT in ACK buffer, Thermo Fisher), cells were collected by centrifugation, resuspended and cultured in B-cell medium (DMEM:IMDM 1:1, Life Technologies), 20% fetal bovine serum (FBS, Sigma-Aldrich), 100 U ml− 1 penicillin/streptomycin (Life technologies), 5 × 10− 5 M 2-mercaptoethanol) supplemented with 0.2 ng ml− 1 murine IL3, 2 ng ml− 1 IL6 and 20 ng ml− 1 SCF (all from Immunotools). Cells were maintained on multi-well plates for suspension cells (Greiner) at ambient oxygen in a humidified cell culture incubator (37 °C with 5% CO2). For colony formation assay, 50,000 cells were plated in MethoCult™ GF M3434 medium (STEMCELL Technologies, 1.5 ml per well on 6-well plate) and colonies were counted after 7 days.
4
Isolation and Characterization of Immune Cells from Ear Tissue
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Freshly isolated ear tissue was diced and subsequently digested with 0.25 mg/ml Liberase TL (Roche) at 37 °C for 135 minutes. Epithelial fragments were removed by filtration (EASYstrainer 70 μm mesh, Greiner Bio-one, Kremsmünster, Austria). Lymph nodes were passed over a 40 μm cell strainer (EASYstrainer, Greiner Bio-one) and washed with PBS. Single-cell suspensions were stained with Live/Dead Fixable Aqua dead cell stain kit (Invitrogen, Waltham, MA) for 20 minutes to exclude dead cells and treated with Fc-blocking anti-mouse CD16/32 antibodies (TruStain FcX; BioLegend). Cells were subsequently stained with fluorochrome-conjugated antibodies against the following surface antigens: CD45, CD3, NK1.1, CD49a, CD49b, CD44, CCR6, CD27, CD11b, CX3CR1, CXCR3, CD127, CD103, IL-23R (all BioLegend), and IL-12Rβ2 (R&D Systems, Minneapolis, MN).
5
Cecal Slurry Preparation for Sepsis Model
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As previously reported, cecal slurry (CS) was prepared (12 (link)). To induce polymicrobial sepsis, male Institute for Cancer Research mice aged 8 to 12 weeks were intraperitoneally injected with CS (13 (link)). After the mice were sacrificed, the cecum was harvested and ground through a 70 μm mesh cell strainer (EASYstrainer™, Greiner Bio-One, Kremsmünster, Austria). The sample was combined with 1–2 mL of sterile phosphate-buffered saline (PBS, Wako, Osaka, Japan) and filtered twice. Following this, the mixture was centrifuged at 11,000 rpm for 1 min. After removing the supernatant, the residue was mixed with 15% glycerol-PBS to achieve a concentration of 500 mg/mL. The sample (400–500 µL) was then transferred to cryogenic biobanking tubes (Greiner Bio-one, Kremsmünster, Austria) and stored at −80°C until use.
6
Isolation and Characterization of Tumor-Associated Macrophages
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After 1 h of tumor digestion using DNAze I (Roche, Basel, Switzerland) and collagenase IV (Collagenase from Clostridium histolyticum; Sigma-Aldrich, Saint-Louis, MO, USA)—both 1 mg/mL) proceeded by mechanical tissue disintegration, the resultant cell suspension was filtered through a 100 µm cell strainer (Easystrainer, Greiner Bio-One, Kremsmünster, Austria). Subsequently, the cells were blocked using an anti-CD16/CD32 blocking antibody (BD Biosciences, San Jose, CA, USA). The cells were then incubated with anti-F4/80 MicroBeads (Miltenyi Biotec Inc., Auburn, CA, USA) and separated using magnetic columns (MACS® Cell Separation Columns—Miltenyi Biotec Inc., Auburn, CA, USA) following the manufacturer’s guidelines. The isolated cells were subsequently subjected to analysis for surface marker expression through flow cytometry or underwent short-term culture (72 h) to purify the material from debris. For the analysis of gene expression via qPCR, lipopolysaccharide (LPS; Sigma-Aldrich, Saint-Louis, MO, USA, 100 ng/mL) was added at a concentration of 100 ng/mL 24 h before lysis in TRI Reagent solution (Molecular Research Center, OH, USA). The cell culture supernatants were utilized for ELISA analysis.
7
Isolation and Cryopreservation of PBMCs
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Monovette blood collection tubes (Sarstedt, Nürnbrecht, Germany) containing ethylenediaminetetraacetic acid (EDTA) were used to collect 36 mL of venous blood. Peripheral blood mononuclear cells (PBMCs) were isolated via a density gradient (Histopaque, 1.077 g/mL, Merck, Darmstadt, Germany). After isolation, PBMCs were counted with a Neubauer improved counting chamber (Laboroptik, Lancing, England). Up to 2×107 PBMCs were resuspended in 1.5 mL cryopreservation medium, consisting of 40 % Roswell Park Memorial Institute 1640 Medium (RPMI) Glutamax (Gibco, Thermo Fisher Scientific, Waltham, USA), 50 % fetal calf serum (FCS, Sigma-Aldrich, St. Louis, USA), and 10 % Dimethyl sulfoxide (DMSO, Sigma-Aldrich). PBMCs were initially frozen at -80 °C and transferred to liquid nitrogen for long-term storage.
Cells were thawed in pre-warmed immune cell medium (ICM), consisting of RPMI Glutamax + 10 % FCS + 50 µg/mL Gentamycin (Gibco). Cells were washed with 10 mL of phosphate-buffered saline (PBS, Gibco), resuspended in 10 mL ICM, and rested for 3 h at 37 °C, 5 % CO2. Thereafter, the cell suspension was passed through a 70-µm cell strainer (EASYstrainer, Greiner) and adjusted to a concentration of 2.5×106 cells per mL of ICM.
Cells were thawed in pre-warmed immune cell medium (ICM), consisting of RPMI Glutamax + 10 % FCS + 50 µg/mL Gentamycin (Gibco). Cells were washed with 10 mL of phosphate-buffered saline (PBS, Gibco), resuspended in 10 mL ICM, and rested for 3 h at 37 °C, 5 % CO2. Thereafter, the cell suspension was passed through a 70-µm cell strainer (EASYstrainer, Greiner) and adjusted to a concentration of 2.5×106 cells per mL of ICM.
8
Isolation and Purification of Splenic Dendritic Cells
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Spleen was collected and dissociated enzymatically using a spleen dissociation kit (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). The spleen was then mechanically dissociated using 40 μm nylon cell EASYstrainer (Greiner bio-one, Dutscher, Brumath, France). Splenic DCs were purified using a positive selection kit (CD11c Microbeads Ultrapure, Miltenyi Biotec GmbH) following the manufacturer’s instructions. Splenic DCs were then maintained in the culture medium at a concentration of 1 × 106 to 2 × 106 cells/mL. The splenic DCs’ purity was assessed by flow cytometry after antibody stanning, as described below, with anti-CD11c-PE-Vio®770 antibody (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). With this method, the degree of purity was between 85% and 95%.
9
Isolation of Naïve CD4+ T Cells
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Mice spleen and lymph nodes were collected and dissociated with 40 μm nylon cell EASYstrainer (Greiner bio-one, Dutscher, Brumath, France). Naïve CD4+ T cells were purified using a naïve CD4+ T cell isolation kit (Miltenyi Biotec GmbH, Bergish Gladbach, Germany), following the manufacturer’s instructions. Naïve CD4+ purity was checked by flow cytometry after staining with anti-CD4-APC antibody (Miltenyi Biotec GmbH, Bergish Gladbach, Germany), and found to be between 85% and 95%.
10
Murine Bone Marrow Isolation and Purification
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For the transplantation experiments, mice were euthanized humanely and both femurs and tibias harvested. These were flushed with sterile PBS, yielding BM cells that were then filtered through a 70-µm EASYstrainer (Greiner). RBCs were lysed using RBC lysis buffer (5 PRIME). For the serial replating and transplantation experiments, BM cells from Ezh2fl/fl; WT or Ezh2fl/fl; Cre+ were selected for cell-surface c-kit expression using CD117 MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol. For the Lin28b/Plag1 in vivo validation experiments, c-kit–positive BM cells from 8–12-wk-old C57/BL6 WT mice were isolated in the same way.
For the ChIP-seq experiments, Lin− HSPCs from whole murine BM were isolated using a Lineage Cell depletion kit (Miltenyi Biotec) as per the manufacturer’s protocol.
For the ChIP-seq experiments, Lin− HSPCs from whole murine BM were isolated using a Lineage Cell depletion kit (Miltenyi Biotec) as per the manufacturer’s protocol.
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