Fresh murine spleen isolated from C57BL/6 mice was torn apart and passed through a 70 µm nylon strainer (BD Falcon) to obtain single cell suspensions. CD4+ and CD8+ T cells were isolated using MagCellect Mouse CD4+ and CD8+ T cell isolation kit (MAGM202 and 203, R&D systems), respectively. For T cell proliferation assay, CD4+ or CD8+ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) and co-cultured with bufalin-primed BMDM (1:1) or bufalin-treated TIM (1:1) in the presence or absence of 2.5 µg/mL anti-CD3 (Invitrogen) and 5 µg/mL anti-CD28 (Invitrogen) for 3 days. CD3, CD4, CD8 and CFSE signals on T cells were detected by flow cytometry and proliferative percentage of CD4+ and CD8+ T cells were calculated by FlowJo software (Tree Star).
70 m nylon strainer
Manufactured by BD
Sourced in United States
The 70 µm nylon strainer is a laboratory equipment designed for filtration and separation purposes. It features a nylon mesh with a pore size of 70 micrometers, allowing for the precise separation and isolation of particles or materials within this size range.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Ack red blood cell lysis buffer, by Lonza (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Tree Star (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by Thermo Fisher Scientific (1 mentions)
Fc block, by BioLegend (1 mentions)
Foxp3 staining buffer kit, by Cytek Biosciences (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Ifn γ xmg1.2, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
11 protocols using 70 m nylon strainer
1
T Cell Proliferation Assay with Bufalin-primed BMDM
2
Tumor Dissociation and Immune Cell Analysis
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Tumors were isolated, rinsed with HBSS, minced with scissors and placed in 3 mL digestion solution containing 100 mg/mL type IV collagenase (Sigma) and 2000 IU/mL type IV DNase (Sigma) in DMEM (Gibco) for 1 hour (37°C, 5% CO2 and humidity) on a plate rocker. Tumor digests were passed through a 70 µm nylon strainer (Falcon) and cells were washed with HBSS (Gibco) and RBCs lysed for 5 min in ACK lysis buffer. Tumor cells were blocked using FC Block (BioLegend) and stained with 1:1000 dilution of TONBO ghost dye for 15 min. Cells were then washed with HBSS containing 2% FBS and stained with 1:200 dilution of antibodies against CD45(30-F11), CD3(17A2), CD4(RM4-5), CD8(53–6.7), CD44(IM7), CD62L(MEL-14), CD25(PC61), CD11b(M1/70), Gr-1(RB6-8C5) (TONBO and BioLegend) for 1 hour at 37°C. Cells were fixed and permeabilized using Foxp3 staining buffer kit (TONBO) then stained for 1 hour at 37°C for granzyme B(QA16A02), IFNγ (XMG1.2) (BioLegend), FoxP3 (MF23) (TONBO). FMO and single stained controls were used for gating and compensation. Gating strategies are indicated within each experiment.
3
Isolation and Culture of Rat Dermal Fibroblasts
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Fibroblast cultures were prepared from the post-natal day-1 Sprague-Dawley rats dorsal skin as described previously with minor modifications [13] . Briefly, each biopsy was minced with sterile scissors and treated with 0.06 mg/ml collagenase I (Sigma, St. Louis, MO) in α-MEM at 37°C for 1.5 h (mixing every 30 min). The digest was then strained into a 50-ml tube containing 5 ml of ice-cold α-MEM supplemented with 20% fetal bovine serum (FBS; Gibco, USA) (termed fibroblast isolation medium) on ice through a 70-µm nylon strainer (BD Falcon, Bedford, MA). Remaining tissue was further digested in 1 mg/ml trypsin (Sigma) at 37°C for 30 min (mixing every 10 min). Digestion was stopped by adding an equal amount of fibroblast isolation medium, and the cells were strained into the same tube. After centrifugation, the cell pellet was resuspended and cultured at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) with Lglutamine (2 mM), 10% fetal bovine serum (FBS; Gibco, USA), penicillin (100 IU/ml), and streptomycin (100 mg/ml) (Thermo, USA). Cells in exponential growth phase between passages 4 and 7 were used for all experiments.
4
Multimodal Flow Cytometric Analysis of 4T1 Tumors
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The 4T1_PB3R-RFP cells (106) in 100 µL of RPMI 1640 medium were injected into the inguinal mammary fat pads of female BALB/c mice. Tumors were allowed to grow to approximately 150 mm3. After systemic treatments, we isolated the tumors from the mice; these tumors were enzymatically digested (Collagenase D, Sigma, USA) into single-cell suspensions and then passed through a 70 µm nylon strainer (BD Biosciences, USA). To analyze the population shift of NAD(P)Hhi (cells high in NAD(P)H intensity), FADhi (cells high in FAD intensity), and RFP+ cells before and after treatment of different therapeutic modalities, we excited endogenous NAD(P)H and FAD signals by applying an ultraviolet laser (375 nm) and a blue laser (488 nm), respectively.23 (link) The NAD(P)H signals were detected using a 450/20 bandpass filter, while the FAD signals were detected using a 530/30 bandpass filter (NAD(P)H emission max=450 nm and FAD emission max=530 nm). To detect RFP signals, we used a red laser (561 nm) with a 585/15 bandpass filter. Flow cytometry standard (FCS) data files were saved and analyzed using FlowJo software V.10.6.2 (BD Biosciences, USA).
5
Isolation of Mammary and Spleen Cells
6
Characterization of hWJSCs by Flow Cytometry
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hWJSCs were disassociated using TrypLETM Express (Invitrogen) for 3–5 min prior to PBS wash to obtain single-cell suspensions, which were then blocked with 10% normal goat serum (NGS) (Invitrogen Life Technologies, Carlsbad, CA) for 30 min to prevent non-specific binding. The cells were incubated with primary antibodies: CD34, CD45, CD73, CD90, and CD105 (1:100, Biolegend, San Diego, CA) for 1 h followed by incubation with Alexa Fluor®488 (1:500) secondary antibody (Invitrogen Life Technologies, Carlsbad, CA) for 30 min. The cells were washed with PBS and re-suspended in 10% NGS. Finally, the cells were filtered using a 70 µm nylon strainer (BD Bioscience) to remove any cell clumps and then analyzed using a CytoFLEX LX Analyzer (Beckman Coulter, Fullerton, CA).
7
Isolation and Characterization of Lung Dendritic Cells and Macrophages
8
Isolation and Characterization of Orbital Fat Stromal Cells
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OASCs were isolated as described previously from patients with and without TAO.7 (link),14 (link) Orbital fat samples were cut into sections less than 5 mm in size. Adipose tissue was digested with 1 mg/mL Col I (Worthington Biochemical Corp, Lakewood, NJ) in Dulbecco's modified Eagle's medium (DMEM) for 3 hours on a shaker. Digested tissue was pipetted up and down at least 10 times before centrifugation at 300g for 5 minutes to remove floating adipocytes. Pellets were suspended in DMEM and filtered with a 70 µm nylon strainer (BD Bioscience, Franklin Lakes, NJ) to yield flow-through cells as stromal vascular fraction. Cells in the stromal vascular fraction were treated with red blood cell lysis buffer to remove red blood cells and with 0.25% trypsin-EDTA for 5 minutes at 37°C to yield a single cell suspension. Cells were maintained in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO2.
9
Antigen-Specific Cytotoxicity Assay
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Splenocytes from female mice were pulsed with 1 µg/ml of the MHC-I-restricted OVA257-264 peptide or HIV-1 gag peptide as a control before labeling with 5 or 0.5 µM of CFSE (Invitrogen, Merelbeke, Belgium), respectively. Labeled cells were mixed at a 1:1 ratio and a total of 1.5 × 107 mixed cells were adoptively transferred into immunized mice 3 days after the second mRNA treatment. Forty eight hours later, splenocytes from mice were isolated and passed through 70 µm nylon strainers (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. Red blood cells were lysed using ACK red blood cell lysis buffer (BioWhittaker, Wakersville, MD, USA). Next, splenocytes were analyzed by flow cytometry. Percentage of antigen-specific killing was determined using the following formula: (1 − (%CFSEhi cells/%CFSElow cells)treated mice/(%CFSEhi cells/% CSFElow cells)non-treated mice) × 100. The experiments were performed on a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA, USA) and analyzed using the FlowJo software (Treestar, OR). Single cells were gated based on their SSC and FSC. Next CFSE-positive cells were selected. See Supplementary Fig. 5 for gating strategy.
10
Induction of Tumor-Specific T-Cell Responses
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C57BL/6 mice were inoculated with 5 × 105 B16 cells, and at days 6 and 10, the mice were treated with saline or 10 µg mRNA encoding Fluc, tBid, or MLKL. Two days after the second treatment, spleens were isolated and passed through 70 µm nylon strainers (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. Red blood cells were lysed using ACK red blood cell lysis buffer (BioWhittaker, Wakersville, MD, USA) and 2.5 × 105 cells were cultured for 24 h in wells of anti-IFN-γ (Diaclone, Besancon, France) pre-coated 96-well plates in the presence of 10 µg/ml peptide. The following synthetic, high-performance liquid chromatography-purified peptides were used for restimulation: OVA 257–264 (SIINFEKL), OVA 323–339 (SQAVHAAHAEINEAGR), CT26-M20 (PLLPFYPPDEALEIGLELNSSALPPTE), CT26-M26 (VILPQAPSGPSYATYLQPAQAQMLTPP), CT26-M03 (DKPLRRNNSYTSYIMAICGMPLDSFRA), CT26-M37 (EVIQTSKYYMRDVIAIESAWLLELAPH), CT26-M27 (EHIHRAGGLFVADAIQVGFGRIGKHFW), B16-M30 mut (PSKPSFQEFVDWENVSPELNSTDQPFL), and B16-M30 WT (PSKPSFQEFVDWEKVSPELNSTDQPFL).
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