Invasion assay was performed as previously reported [18 (link)]. Briefly, the cells treated with either inhibitors or siRNAs for transfection were seeded in the upper well of a Transwell chamber (8-μm pore size) that was pre–coated with 10 mg/mL growth factor-reduced Matrigel (BD Bioscience). After 72 h, non–invading cells on the upper surface of the filter were removed with a cotton swab, and the migrated cells on the lower surface of the filter were fixed and stained with a Kwik-Diff kit (Thermo Fisher Scientific, Waltham, MA, USA). Invasiveness was determined by counting cells in fields per well, and the extent of invasion was expressed as the average number of cells per microscopic field. The cells were imaged by phase contrast microscopy. For the migration assay, we used Transwell chambers with inserts that contained the same type of membrane but without the Matrigel coating.
Kwik diff kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Kwik-Diff™ kit is a rapid staining solution used for the differentiation of blood cells in microscopy applications. The product provides a fast and reliable method for staining and preparing samples for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hemocytometer, by Hausser Scientific (2 mentions)
Cytospin 4 centrifuge, by Thermo Fisher Scientific (2 mentions)
Axio imager m1, by Zeiss (2 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Transwell chamber, by BD (1 mentions)
Transwell insert, by Corning (1 mentions)
Dy998, by R&D Systems (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Hematoxylin qs, by Vector Laboratories (1 mentions)
Biocoat matrigel invasion chamber, by Corning (1 mentions)
8 protocols using kwik diff kit
1
Transwell-Based Cell Invasion Assay
2
BALF Cell Differential Quantification
3
Cell Migration and Invasion Assay
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Migration: Cells were seeded into 24 wells at a density 2 × 105 cells/well with serum-free media in the upper chamber (0.8 μm pore size, Corning, Corning, NY, USA) and media containing 10% FBS in the lower chamber. Invasion: Cells were plated in serum-free medium on transwell inserts (Corning, NY, USA) coated with 5 μg of matrigel.
After 24 h incubation, cells that had migrated or invaded into the lower surface of the insert were fixed and stained using Kwik-Diff kit (Shandon, Thermo Scientific, Waltham, MA, USA). The number of migrating/invasive cells was counted in five representatives fields per insert at ×100 magnification.
After 24 h incubation, cells that had migrated or invaded into the lower surface of the insert were fixed and stained using Kwik-Diff kit (Shandon, Thermo Scientific, Waltham, MA, USA). The number of migrating/invasive cells was counted in five representatives fields per insert at ×100 magnification.
4
BALF Cell Differential Quantification
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Collected fluid was processed for total and differential cell counts by the Duke Rodent Inhalation core, which was blinded to the condition. Collected fluid was centrifuged at 3000 rpm for 10 mins at 4C, cells were treated with 1XRBS lysis buffer, further centrifuged, and resuspended in PBS. Cells were counted with a hemocytometer (Hausser Scientific, Horsham, PA), and recovery volume was used to determine cell density. BALF cytology was performed by immobilizing 100uL of the cell suspension using a Cytospin 4 centrifuge (Thermo Fisher Scientific, Waltham, MA). Cells were stained with a Kwik-Diff™ kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer recommendations. Images were obtained using a 20x objective on an AxioImager M1 (Zeiss) microscope. Cell differential counts were determined by morphological analysis of acquired images and were used to identify and quantify macrophages, neutrophils, lymphocytes, and eosinophils. Out of the <2,000 cells counted, less than 5 were eosinophils. Thus eosinophils were excluded from further analyses.
5
Allergic Inflammation Biomarker Quantification
6
Quantifying Lung Biopsy Inflammation
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Biopsies were obtained from explanted lungs, fixed in formalin, and embedded in paraffin. Sections were deparaffinized and stained following standard procedures. Slides were stained with GDF15 antibodies (G-5, Santa Cruz Biotechnology), developed with 3, 3′-diaminobenzidine, and counterstained with hematoxylin QS, according to the manufacturer’s protocol (Vector Laboratories). Slides were scanned at HistoWiz. Differential counts were performed on BAL cells that had been spun onto slides and stained with Kwik-Diff kit, according to the manufacturer’s protocol (Thermo Fisher Scientific).
7
Astrocytic Differentiation in Glioblastoma
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The different concentrations of MGCD0103 were used to identify the ability of mocetinostat to induce astrocytic differentiation in glioblastoma cell lines C6 and T98G. Using 6 well plates, cells were cultivated in growth medium RPMI-1406 with 10% FBS and 1% Penicillin/Streptomycin. The cultures were placed in the incubation with 5.0% CO2 and 37 C for 24h. After the culture medium was removed, the cells cultured in the medium were supplemented with different concentrations of MGCD0103. The cells were placed in the incubation conditions of 5% CO2 at 37 C for 48h. After treating the cells with drugs, the culture medium was aspirated and washed twice with BPS. After that, the cells were fixed and stained using a Kwik-Diff™ kit according to the protocol of the manufacturer (Thermo-Scientific, MO, USA). The samples were examined under light microscopy to watch the differentiation features of cells.
8
Cell Invasion Assay with Matrigel
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BioCoat™ Matrigel™ Invasion Chambers (Corning™, Thermo Fisher Scientific, cat. no. 08-774) were plated with 50,000 cells (upper chamber) in triplicate, and incubated under standard conditions for 24 h. The media from the upper chamber then was removed, and any cells remaining in the upper chamber were removed using a cotton swab. Cells that had migrated to the bottom of the membrane were stained using a Kwik-Diff™ kit (Shandon™, Thermo Fisher Scientific, cat. no. 9990701). Membranes were mounted onto glass slides, and cells per highpower field were counted using ImageJ software.
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