Cells were cultured on culture dishes (for immunoprecipitation), coverslips (for immunostaining of A431 cells), or 12-well polycarbonate Transwell filters with 0.4-µm pore size (#3401; Corning) at 1.0 × 105 cells/ml (for immunostaining of MDCK II cells). Cells were washed with FBS-free DMEM thrice at 37°C, treated with Trypsin (0.25% [wt/vol] Trypsin-1 mM EDTA•4Na solution; #209-16941; Fujifilm-WAKO) diluted with FBS-free DMEM, and incubated at 37°C for 15 min in a 5% CO2 incubator. As EDTA was diluted more than 10 times with DMEM, which contains 1.8 mM CaCl2 and 1 mM MgCl2, it did not affect the cell adhesion. The cells were then washed and processed for subsequent applications.
Trypsin
Manufactured by Fujifilm
Sourced in Japan, United States
Trypsin is a proteolytic enzyme that cleaves peptide bonds in proteins. It is commonly used in cell culture and biochemistry applications to dissociate adherent cells from tissue culture surfaces and to facilitate the extraction and processing of proteins from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Fujifilm (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Lys c, by Fujifilm (5 mentions)
Collagenase, by Fujifilm (5 mentions)
Lysyl endopeptidase, by Fujifilm (4 mentions)
Trypsin, by Promega (4 mentions)
Collagen 1, by Iwaki (3 mentions)
Hank s balanced salt solution, by Fujifilm (3 mentions)
Collagenase type 2, by Worthington (3 mentions)
Fetal bovine serum (fbs), by Biowest (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Streptomycin, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Edta 4na solution with phenol red, by Fujifilm (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Apm 30d, by Astec (2 mentions)
L glutamine, by Fujifilm (2 mentions)
Monospin c18 column, by GL Sciences (2 mentions)
Trypsin inhibitor, by Fujifilm (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
88 protocols using trypsin
1
Trypsinization and Cell Preparation
2
Primary Culture of Mouse Astrocytes
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The animal experimentation protocol in the present study was approved by the Animal Care and Experimentation Committee, Gunma University (19-024, 17 December 2018), and all efforts were made to minimize animal suffering and the number of animals used.
A primary culture of mouse cerebral cortex astrocytes was prepared as previously described (29 (link), 30 (link)) with slight modifications. A pregnant C57BL/6 strain mice were purchased from Japan SLC (Hamamatsu, Japan). Briefly, postnatal day 1 mouse cerebral cortices were dissected and digested with 2.5% trypsin (Wako, Japan) in Hank’s balanced salt solution (Wako) for 30 min with continued shaking at 37°C. Cells were resuspended in an astrocyte culture medium (high-glucose DMEM, 10% heat-inactivated FBS, and 1% penicillin/streptomycin), and 10–15 million cells were plated on 10-cm dishes coated with Collagen I (Iwaki, Japan). Cells were incubated at 37°C in a CO2 incubator. On day 3 in vitro (DIV3), astrocyte culture medium was replaced with phosphate-buffered saline (PBS). Dishes were then shaken by hand for 30–60 s until only the adherent monolayer of astrocytes was left. The PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 using 0.25% trypsin 1 mM disodium EDTA (Wako), and then plated on 12 or 24 well dishes. Cells were used for cell invasion assay or F-actin staining.
A primary culture of mouse cerebral cortex astrocytes was prepared as previously described (29 (link), 30 (link)) with slight modifications. A pregnant C57BL/6 strain mice were purchased from Japan SLC (Hamamatsu, Japan). Briefly, postnatal day 1 mouse cerebral cortices were dissected and digested with 2.5% trypsin (Wako, Japan) in Hank’s balanced salt solution (Wako) for 30 min with continued shaking at 37°C. Cells were resuspended in an astrocyte culture medium (high-glucose DMEM, 10% heat-inactivated FBS, and 1% penicillin/streptomycin), and 10–15 million cells were plated on 10-cm dishes coated with Collagen I (Iwaki, Japan). Cells were incubated at 37°C in a CO2 incubator. On day 3 in vitro (DIV3), astrocyte culture medium was replaced with phosphate-buffered saline (PBS). Dishes were then shaken by hand for 30–60 s until only the adherent monolayer of astrocytes was left. The PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 using 0.25% trypsin 1 mM disodium EDTA (Wako), and then plated on 12 or 24 well dishes. Cells were used for cell invasion assay or F-actin staining.
3
Primary Culture of Mouse Astrocytes
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Primary culture of mouse cerebral cortex astrocytes was prepared as previously described44 (link),57 . Pregnant C57BL/6 strain mice were purchased from Japan SLC (Hamamatsu, Japan). Briefly, postnatal day 1, mouse cerebral cortices were dissected and digested with 2.5% trypsin (Wako, Japan) in Hank's balanced salt solution (Wako) for 30 min with continuous shaking at 37 °C. Cells were resuspended in an astrocyte culture medium (high-glucose Dulbecco's Modified Eagle Medium, 10% heat-inactivated fetal bovine serum, and 1% penicillin/streptomycin), and 10–15 million cells were plated on 10-cm dishes coated with Collagen I (Iwaki, Japan). Cells were incubated at 37 °C in a CO2 incubator. On day 3 in vitro (DIV3), the astrocyte culture medium was replaced with phosphate-buffered saline (PBS). Dishes were then shaken by hand for 30–60 s until only the adherent monolayer of astrocytes was left. The PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 using 0.25% trypsin 1 mM disodium EDTA (Wako) and then plated on 12 or 24 well dishes. Cells were used for cell invasion assay or F-actin staining.
4
Oral Cancer Cell Line HSC-3 Cultivation
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In this study, we obtained an oral cancer cell line, HSC-3, from the Health Science Research Resources Bank (Osaka, Japan). This cell line originates from a metastatic deposit of a poorly differentiated squamous cell carcinoma (SCC) of the tongue, specifically in a mid-internal jugular lymph node, extracted from a 64-year-old man. The cultivation of HSC-3 cells was performed using Eagle’s minimum essential medium (Sigma–Aldrich, St. Louis, MO, USA), which was supplemented with fetal bovine serum (10%; Thermo Fisher Scientific, Waltham, MA, USA), as well as a solution of penicillin and streptomycin (1000 units/mL; Sigma–Aldrich). Trypsin (0.25%; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and ethylenediaminetetraacetic acid (0.02%; DOJINDO LABORATORIES, Kumamoto, Japan) solutions were used to isolate cells for subculture, as previously described [24 (link)].
5
MALDI-TOF Mass Spectrometry Protocol
6
RNA Extraction from HEK293T Cells
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HEK293T/17 cells were washed with D-PBS (-) (Nacalai tesque) and detached using 0.05% w/v Trypsin (Wako). Cells were collected by centrifugation for 5 mins (6000 × g) at 4°C and total RNAs were subsequently extracted using an RNeasy Mini Kit (QIAGEN) following the manufacturer’s protocol. Samples were treated 3 times with DNase I to digest transfected plasmid DNA with the TURBO DNA-free kit (Thermo Fisher Scientific). RIN values and gel band intensities of the total RNA were checked using the Agilent 2100 Bioanalyzer (Agilent Technologies) following the Agilent RNA 6000 Nano kit protocol. The concentration of total RNA and absorbance at 230/260 and 260/280 were measured by NanoDrop8000 (Thermo Fisher Scientific).
7
Cell Counting of Adherent Cell Lines
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Following treatment, the media used for culturing HL60 cells was gently mixed 15 times with a dropper, and the floating and weakly bound cells suspended in the medium were counted using a MOXI Z automated cell counter (ORFLO, Ketchum, ID, USA). Moderately and tightly bound cells, which were resistant to initial gentle agitation, were treated with 0.05% w/v trypsin–0.53 mmol/L EDTA (FUJIFILM Wako Pure Chemical Co.) and detached by pipetting for adherent cell counts.
The MG63 and Balb/c cells tightly adhered to the bottom surface of the dish. The cells were not detached using gentle agitation. Thus, the culture media was aspirated without agitation, and the cells were enzymatically detached for cell counting, as described above.
The MG63 and Balb/c cells tightly adhered to the bottom surface of the dish. The cells were not detached using gentle agitation. Thus, the culture media was aspirated without agitation, and the cells were enzymatically detached for cell counting, as described above.
8
Nef Internalization and Macropinocytosis in Macrophages
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The internalization of Nef into MΦ was quantified by flow cytometry on an LSR II (BD Biosciences) using the FlowJo software (Tree Star, Ashland, OR, USA). MΦ were incubated with FITC-labeled Nef (purchased from Fitzgerald, Acton, MA, USA) at 37 °C, detached from the wells using 0.25% trypsin (Wako) after extensive washing with PBS, and immediately subjected to flow cytometric analysis.14 (link) The macropinocytosis activity of MΦ was analyzed by measuring the uptake of lucifer yellow.36 (link) MΦ were incubated with lucifer yellow CH dipotassium salt (Sigma; 100 μg/ml) for 2, 4, or 6 h, detached from the wells using 0.25% trypsin after extensive washing with PBS, and immediately subjected to flow cytometric analysis. The surface expression of CD163 on MΦ and M-CSF receptor on RAW264.7 cells was also assessed by flow cytometry. Cells left untreated or stimulated with Nef were detached from the wells using enzyme-free cell dissociation buffer (Gibco, Grand Island, NY, USA), stained with PE-labeled anti-human CD163 (GHI/61; BioLegend, San Diego, CA, USA), or PE-labeled anti-mouse M-CSF receptor (AFS98; eBioscience, San Diego, CA, USA) and analyzed on the LSR II using the FlowJo software.
9
Primary Culture of Mouse Cerebellar Astrocytes
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A primary culture of mouse cerebellar astrocytes was prepared as previously described [51 (link),52 (link)] with slight modifications. Briefly, P1 mouse cerebella were dissected and digested with 2.5% trypsin (Wako, Osaka, Japan) in HBBS (Wako) for 30 min with continued shaking at 37 °C. The cells were resuspended in an astrocyte culture medium (DMEM high-glucose, 10% heat-inactivated FBS, 1% penicillin/streptomycin), and 10–15 million cells were plated on 10 cm dishes coated with Collagen-I (Iwaki, Tokyo, Japan). The cells were incubated at 37 °C in the CO2 incubator. On DIV3, astrocyte culture medium was replaced with PBS. Dishes were then shaken by hand for 0.5–1 min until only the adherent monolayer of astrocytes was left. PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 with 0.25% trypsin 1 mmol/L Na.EDTA (Wako), and then plated on 12- or 24-well dishes. The quality of primary culture of cerebellar astrocyte was examined by immunocytochemistry with astrocyte marker including S100β and GFAP (Supplementary Figure S3 ). The cells were used for cell proliferation, invasion or F-actin activity assays.
10
Isolation and Characterization of Stromal Cells
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The tissues were treated with RPMI-1640 (Wako) containing 0.1% BSA, 1 mg/ml Collagenase type Ι (Wako), and 2 μg/ml DNase Ι (Sigma) for 45 min at 37 °C, and the cell suspension was overlaid on 40% FCS (fetal calf serum; PAA Laboratories) for 1 h. The lower stroma cell fraction was collected and treated with 0.2% trypsin and 0.5 mM EDTA (Wako) for 15 min at 37 °C, subsequently with 10 μg/ml DNase Ι and 10% FCS. A single cell suspension passed through a nylon mesh with 108 μm pore size were then treated with culture supernatant containing anti-CD16/32 antibody, followed by FITC-labeled anti-CD45, biotin-labeled MECA-79, biotin-labeled MECA-89, and eFluor660-labeled CD34 mAbs, PE-labeled streptavidin, and 7-aminoactinomycin D (7-AAD). The data analysis and interpretation were carried out using a FACS Aria ΙΙ and the FlowJo software.
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