MEFs or primary macrophages (5 × 103) were added to a cell culture insert (8 μm, Greiner Bio-one). After incubation for 24 hours, the cells were stained using Diff-quick staining kit (Sysmex). Nonmigrated cells on the upper surface were scraped off. Areas of migrated cells on the lower surface were measured using Image J software.
Diff quick staining kit
Manufactured by Sysmex
Sourced in Japan
The Diff-Quick staining kit is a set of ready-to-use solutions used for the rapid staining of blood smears and other cytological preparations. The kit provides a simple and efficient method for differentiating and identifying different types of blood cells.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Cell culture insert, by Greiner (1 mentions)
Matrigel coating, by Corning (1 mentions)
Matrigel coated, by BD (1 mentions)
Boyden chamber, by BD (1 mentions)
Cell culture insert, by BD (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Transwell assay, by BD (1 mentions)
Histofine simple stain kit, by Nichirei Biosciences (1 mentions)
Wst 8 cell counting kit, by Fujifilm (1 mentions)
Matrigel invasion chamber, by BD (1 mentions)
Transwell chamber, by Corning (1 mentions)
Transwell insert, by Corning (1 mentions)
Matrigel coated transwell insert, by BD (1 mentions)
13 protocols using diff quick staining kit
1
Quantifying Cell Migration Assay
2
Invasion Assay for Cancer Cells
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Invasion assays were performed using 24‐well plates with 8 μm membrane pores and a Matrigel coating (#354480, Corning, NY). We overlaid 1.0 × 105 (MiaPaCa2, BxPC3) or 5.0 × 104 (PSN1) cells onto the Matrigel matrix on a membrane with 8‐mm diameter pores. After 48 h, the invasive cells growing at the bottom of the membrane were fixed with methanol and stained using the Diff‐Quick staining kit (16 920, Sysmex, Hyogo, Japan). Three microscopic fields were randomly selected for cell counting.
3
Boyden Chamber Cell Invasion Assay
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For Boyden chamber-based cell invasion assay, tumor cells (0.5×104) suspended in 0.5 ml of RPMI-1640 containing 0.1% FCS were plated on Matrigel-coated 8-µm pore diameter polypropylene filter inserts in the Boyden chamber (BD Biosciences). Cells were allowed to migrate for 24 h toward 1 ml of RPMI-1640 containing 10% FCS as chemoattractant. Cells remaining in the insert were removed with a cotton swab, and the cells which attached to the bottom of the filter were stained with Diff-Quick-Staining kit (Sysmex, Kobe, Japan) and counted under optical microscope. At least five fields were counted in each experiment. For assays in the presence of antibodies, cells were pretreated with antibody at the indicated concentration for 15 min at room temperature. For Boyden chamber-based cell motility assay, uncoated inserts (8-µm pore diameter) was used.
4
Colony Forming Assay Protocol
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The plasma-exposed cells were plated in 100-mm cell culture dishes (400 cells/dish) and incubated for 2 weeks at 37°C in an incubator. The medium was changed every 3 days. The colonies were stained using the Diff-Quick staining kit (Sysmex, Kobe, Japan) according to the manufacturer's instructions. The colony forming units (CFU) were considered visible. The images of visualized CFU were taken and counted using image J software.
5
Colony Formation Assay with 5-FU
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Cells were seeded at a density of 500 cells per 60 mm dish and cultured for 10 days with either PBS as a control or with 9 μM 5-FU in SNUC5 cell and 2264 μM 5-FU in SNUC5/5-FUR cells. During colony growth, the culture medium was replaced every 3 days. Colonies with more than 50 cells were counted under microscopic observation using a Diff-Quick staining kit (Sysmex, Kobe, Japan).
6
Cell Migration Assay Using Transwell
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Cell culture inserts (8-mm pore size and 6 mm in diameter; BD Biosciences, Franklin Lakes, NJ) were used following the manufacturer's instructions. Briefly, 1 Â 10 5 NV-CML cells were suspended in 500 mL serum-free medium and placed onto the upper component of the inserts. The lower compartment was filled with 750 mL of a medium containing 10% FBS, and the cells were incubated at 37 C in a humidified 5% CO 2 atmosphere. After 24 hours, the cells on the upper surface of the filter were carefully removed with a cotton swab. The cells that had migrated through the membrane to the lower surface of the filter were fixed and stained with a Diff-Quick staining kit (Sysmex Corp, Kobe, Japan). The number of cells on each membrane was counted in 10 high-power (Â200) fields spaced evenly over the whole area of the round membrane, and the mean value was calculated per field.
7
Transwell Assay for Evaluating Cell Invasion
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The invasive ability of the cultured cells was assessed using the Transwell assay and BD Biocoat Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) according to the manufacturer’s instructions. In brief, 1 × 105 cells were plated in the upper chamber in serum-free DMEM, whereas the medium in the bottom chamber contained 10% FBS, and the plates were incubated for 48 h. The tumor cells invading the bottom surface were stained with Diff-Quick staining kit (Sysmex, Hyogo, Japan) according to the instruction manual, and their number was counted in nine separate areas under a microscope at the magnification of 200×.
8
Nestin Phosphorylation and Cell Dynamics
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Reagents were purchased from the following companies: mouse monoclonal anti‐nestin antibody from R&D Systems, Inc. (Minneapolis, MN, USA); rabbit polyclonal anti‐Thr315 and anti‐Thr1299 phosphorylated nestin antibodies from Santa Cruz Biotechnology (p‐nestin, sc‐33879 for phosphorylated‐Thr315 of human nestin, sc‐33880 for phosphorylated‐Thr1299 of human nestin; Santa Cruz, CA, USA); rat monoclonal anti‐phosphorylated Histone H3 (phospho S28, ab10543) from Abcam (Cambridge, UK); mouse monoclonal anti‐MIB‐1 antibody from Dako Denmark A/S (Glostrup, Denmark); Histofine Simple Stain kit from Nichirei (Tokyo, Japan); Zenon rabbit IgG labeling kit (Z‐25351), Hoechst 33342, and Click‐iT EdU Pacific Blue Flow Cytometry Assay Kit from Invitrogen (Carlsbad, CA, USA); DSRed‐Express2‐N1 vector from Clontech (Mountain View, CA, USA); FuGene HD transfection reagent from Roche Diagnostics (Mannheim, Germany); the WST‐8 Cell Counting Kit from Wako Pure Chemical Industries (Osaka, Japan); Matrigel invasion chambers from BD Bioscience (Franklin Lakes, NJ, USA); Diff‐Quick staining kit from Sysmex Corp. (Kobe, Japan).
9
Transwell Invasion Assay for SKOV3 Cells
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Invasion assays were performed using a 6.5 mm Transwell chamber with an 8.0 μm pore filter (Costar; Cambridge, MA, USA). Each Transwell plate was coated with 1 mg/mL Matrigel (BD Bio Sciences; San Jose, CA, USA), and 5 × 104 SKOV3 cells were seeded into the upper chamber and maintained at 37 °C for 48 h. After incubation, the cells remaining on the upper surface of the chamber were removed using a cotton swab, and the cells that had moved to the lower chamber were fixed and stained with a Diff-Quick staining kit (Sysmex; Kobe, Japan) according to the manufacturer’s protocol. Briefly, invading cells migrated and attached to the bottom of the membrane, and invaded cells were fixed with methanol, cytosol was stained with eosin Y, and the nucleus was stained with methylene blue. An inverted microscope at 100× magnification was used to image the stained cells in five random fields, and the number of cells in each field was manually counted.
10
Colony Formation Assay for SW480 Cells
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SW480 cells were seeded at a density of 500 cells per 60 mm dish in triplicate and cultured for 10 days with either PBS (as a negative control) or 40 μg/mL DUE. During colony growth, the culture medium was replaced every 3 days. Colonies with more than 50 cells were counted. The cells were counted under microscopic observation using a Diff-Quick staining kit (Sysmex, Kobe, Japan).
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