HL-60 cells (2×106/ml) were treated with 0.5% DMSO or PTER (0∼100 µM) for 24 h. At the end of incubation, cells were collected and fixed with 70% ethanol. Cells were stained with propidium iodide (PI) buffer (4 µg/ml PI, 1% Triton X-100, and 0.5 mg/ml RNase A in phosphate-buffered saline (PBS)) for 30 min in the dark at room temperature and then filtered through a 40-µm nylon filter (Falcon, San Jose, CA). The cell cycle distribution was analyzed for 10,000 collected cells by a FACS Vantage flow cytometer that uses the Cellquest acquisition and analysis program (Becton-Dickinson FACS Calibur, San Jose, CA). The proportion of nuclei in each phase of the cell cycle was determined, and apoptotic cells with hypodiploid DNA content were detected in the sub-G1 region. All results were obtained from three independent experiments.
40 m nylon filter
Manufactured by BD
Sourced in United States
The 40-μm nylon filter is a laboratory equipment designed for filtration purposes. It is made of nylon material and has a pore size of 40 micrometers. The filter's core function is to separate particles or substances of a specific size from a liquid or gas sample.
Automatically generated - may contain errors
Lab products found in correlation
Facsvantage, by BD (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Rbc lysing buffer, by BioLegend (1 mentions)
Anti cd3ε 145 2c11, by BioXCell (1 mentions)
Anti cd28 antibodies, by BioXCell (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Cell strainer, by BD (1 mentions)
Rneasy plus universal kit, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
5 protocols using 40 m nylon filter
1
Cell Cycle Analysis of HL-60 Cells
2
Analyzing Tumor-Infiltrating T Cells
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For preparation of single cell suspensions, tumor tissues were mechanically dissected and washed with PBS. The cell suspension was passed through a sterile 40‐µm nylon filter (BD Falcon). A single‐cell suspension was obtained, and 0.5‐1 × 106 cells were collected and stained with PerCP‐anti–mouse‐CD4 and APC‐anti–mouse‐CD8α antibodies to detect the percentage of CD4+ and CD8+ T cell tumor infiltration. Next, to further examine tumor infiltrating T cell exhaustion, the cell suspensions continued to be stained by PE‐anti–mouse‐PD‐1, PE‐anti–mouse‐LAG‐3, or PE‐anti–mouse‐Tim‐3 antibodies, respectively. Finally, these stained cells were analyzed on the FACSCanto II Flow Cytometer (BD Biosciences), and FACS data were analyzed with FlowJo software (TreeStar).
3
Murine T Cell Isolation and Culture
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The spleens of 6‐to 8‐week‐old C1QBP+/− and C1QBP+/+ C57/BL6 mice were harvested, gently ground in MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) and then passed through a sterile 40‐µm nylon filter (BD Falcon). Red blood cells were lysed with RBC Lysing Buffer (BioLegend). The murine T cells were isolated by Pan T magnetic Microbeads (Miltenyi Biotec) from splenocytes obtained from C1QBP+/+ and C1QBP+/− mice. CD4+ or CD8+ T cells were sorted on a FACSAria III Cell Sorter (BD Biosciences) and planted in 24‐well plates coated with 2.5 μg/mL anti–CD3ε (145‐2C11, Bio‐X‐Cell) and 1 μg/mL anti–CD28 antibodies (37.51 Bio‐X‐Cell). All the above cells were cultured in RPMI‐1640 medium (Corning) supplemented with 10% FBS, 4 mM l ‐glutamine, 1% penicillin/streptomycin, 2 ng/mL IL‐2, 10 ng/mL IL‐7, 5 ng/mL IL‐15, and 75 μM β‐mercaptoethanol and incubated at 37°C in a 5% CO2 humidified atmosphere.
4
Cell Cycle Analysis of Glabridin Treatment
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To determine the effect of glabridin on cell cycle, cells (1×106/ml) were first cultured in serum-free medium for starvation at 18 hours and then exposed to glabridin for 24 hours. After cells were washed, fixed with 70% ethanol, and incubated for 30 minutes in the dark at room temperature with propidium iodide (PI) buffer (4 µg/ml PI, 1% Triton X-100, 0.5 mg/ml RNase A in PBS) and then filtered through a 40-µm nylon filter (Falcon, USA) [16] (link). The cell cycle distribution was analyzed for 5,000 collected cells by a FACS Vantage flow cytometer that uses the CellQuest acquisition and analysis program (Becton Dickinson FACSCalibur).
5
Transcriptomic Insights into Temperature-Dependent Longevity
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To gain molecular insights into the NPR-8-dependent longevity response to temperature, we employed RNA-seq to compare gene expression in young and old wild-type and npr-8(ok1439) animals propagated at different temperatures. To this end, we collected five replicates of eight groups of RNA samples (young (0-day-old) and 9-day-old adult wild-type and npr-8(ok1439) animals propagated at 20°C and 25°C) (Table 1). The temperature-specific worm cultivation and synchronization steps were performed as described in the lifespan assay section. A portion of synchronized adult animals were collected at the age of 0-day old, and the remaining animals were grown till day 9 before collection. These animals were washed every day using M9 buffer and the adult animals were filtered using cell strainer (40-µm nylon filter, Falcon) and transferred to E. coli OP50-seeded NGM plates. Worms were washed and collected with M9 buffer then snap-frozen in the TRIzol reagent (Thermo Fisher Scientific) and stored at -80°C until RNA isolation. RNA was extracted using the QIAzol lysis reagent (Qiagen) and purified with the RNeasy Plus Universal Kit (Qiagen).
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