Migration assays were performed in cell culture inserts (24-well, 8-µm pore size, #353097, Corning, Inc.). Cell concentrations ranged from 105 to 2×105 cells/ml. Invasion assays were also performed in Corning BioCoat Matrigel invasion chambers (24-well, 8-µm pore size, #353097, Corning, Inc.). Cell concentrations ranged from 2×105 to 4×105 cells/ml. Cells were seeded on uncoated or Matrigel-coated inserts in 500 ml of serum-free medium for migration and invasion assays, respectively. The lower chambers were filled with 750 µl of 10% FBS-supplemented medium. After 48 h, the cells on the lower surface of the insert were fixed and stained with crystal violet (Differential Quik Stain kit, Polysciences) at room temperature for 2 min. The number of stained cells was counted in >3 fields under an inverted microscope (Olympus CKX31).
Differential quik stain kit
Manufactured by Polysciences
Sourced in United States
The Differential Quik Stain Kit is a laboratory staining solution designed for the differentiation and identification of cellular components in various sample types. It provides a rapid and reliable method for staining and visualizing cellular structures under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel basement membrane matrix, by Corning (2 mentions)
Permeable support, by Corning (2 mentions)
Cellsens standard software, by Olympus (2 mentions)
Bx43 microscope, by Olympus (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Ckx31, by Olympus (1 mentions)
Biocoat matrigel invasion chamber, by Corning (1 mentions)
Ix73 microscope, by Olympus (1 mentions)
Biocoat invasion chamber, by BD (1 mentions)
Biocoat growth factor reduced matrigel invasion chamber, by BD (1 mentions)
Axiovert 25, by Zeiss (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Fluorchem r imager, by Bio-Techne (1 mentions)
Western lightning ecl pro, by PerkinElmer (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Abcam (1 mentions)
Anti β actin antibody 8h10d10, by Cell Signaling Technology (1 mentions)
Polyethylenimine max pei max, by Polysciences (1 mentions)
Pires2 acgfp1 vector, by Takara Bio (1 mentions)
18 protocols using differential quik stain kit
1
Cell Migration and Invasion Assay Protocol
2
Neutrophil Enumeration in BALF Samples
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The total number of cells in each BALF sample was counted and samples were adjusted to 750,000 cells per ml. 200,000 cells per sample were added to the cytospin apparatus and centrifuged onto glass slides at 700 rpm for 5 minutes at room temperature. Slides were dried and stained with the Differential Quik Stain Kit (Polysciences, Warrington, PA) according to the included protocol. Neutrophils were enumerated under 100x magnification using an Olympus IX-73 microscope. Original neutrophil numbers per ml of BALF were calculated by accounting for the dilution factors used to adjust cells to 750,000/ml.
3
Invasion Assay for miR-135b
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The invasion assay was performed using a 24-well BD BioCoat Invasion Chamber (BD Biosciences, USA). Cells transfected with 10nM of mirVana inhibitor or 15nM of mirVana mimic to miR-135b and appropriate controls were harvested 24 hours after transfection. The cells were re-suspended in a serum free media with reduced growth factors (as previously stated) and added to the upper chamber at a density of 1x104 cells per well in triplicate; the bottom chamber contained growth media supplemented with 10% fetal bovine serum (FBS) and normal concentration of growth factors. After incubating for 24 hours, cells in the upper well were removed by aspiration of the Matrigel and wiping the top of the membrane with a cotton swab. Cells on the underside of the membrane were fixed using the Differential Quik Stain Kit (Polysciences, Inc., USA). Briefly, the membranes were submerged sequentially in each solution (A, B, and C) for 2 minutes, then rinsed twice in deionized water and allowed to air dry. Invading cells were evaluated using images of five microscopic fields per membrane under light microscopy at a magnification of 40x. The number of invading cells in each photograph were then counted using NIH Image J program 1.48b.
4
Cell Invasion Assay with Matrigel
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The experiment was performed as described in Corning’s Cell Invasion Assay protocol (available online at the following URL: http://csmedia2.corning.com/LifeSciences/media/pdf/protocol_DL_031_Cell_Invasion_Assay.pdf ). Briefly, permeable supports (Corning, cat. #353097) were coated with 200 μg/ml Matrigel basement membrane matrix (Corning, cat. #354234), and inserted in 24-well companion plates (Corning, cat. #353504). Cell suspensions were prepared in serum-free DMEM, and seeded in invasion chambers at 25,000 cells/chamber. DMEM with 20% FBS was added to each well as chemoattractant. Samples were incubated at 37°C for 24 hours to allow for cell migration through the Matrigel. Non-invading cells on the apical surface of the Matrigel-coated supports were removed with cotton swabs, and cells that had migrated to the lower surface of the supports were stained using Differential Quik Stain Kit (Polysciences, cat. #24606). Photographs were taken, and cells were counted using ImageJ. Samples were prepared in triplicate, and 5 fields were photographed per sample (at 20X magnification).
5
Matrigel Invasion Assay
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Either 48 h after 4-OHT treatment, or 72 h after siRNA treatment/lentiviral expression, cells were detached with Accutase and counted. Sets of 5x 10
4 cells were spread onto the top chamber of BDBioCoat growth factor reduced MATRIGEL invasion chambers (BD). Assays were performed according to manufacturer’s protocol, by using 5% horse serum and 20 ng/ml EGF as chemoattractants. Positive invading cells were stained with Differential Quik Stain kit (Polysciences) and counted from ten independent fields at 20x magnification (Zeiss Axiovert 25).
4 cells were spread onto the top chamber of BDBioCoat growth factor reduced MATRIGEL invasion chambers (BD). Assays were performed according to manufacturer’s protocol, by using 5% horse serum and 20 ng/ml EGF as chemoattractants. Positive invading cells were stained with Differential Quik Stain kit (Polysciences) and counted from ten independent fields at 20x magnification (Zeiss Axiovert 25).
6
Measuring Toxoplasma gondii Plaquing and Growth
7
Optimized Cell Culture Reagents and Protocols
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DMEM and RPMI1640 were obtained from FUJIFILM Wako Pure Chemical Industries (Osaka, Japan). Fetal bovine serum (FBS), anti-Xpress antibody (catalog number; 46-0528), SuperScript IV Reverse Transcriptase, Lipofectamine 3000, antibiotic–antimycotic and pcDNA4/His B vector were from Thermo Fisher Scientific (Waltham, MA, USA). pIRES2-AcGFP1 vector was from Takara Bio (Kusatsu, Japan). KOD-Plus DNA polymerase was from Toyobo (Osaka, Japan). SV Total RNA Isolation System was from Promega KK (Tokyo, Japan). Western Lightning ECL Pro was from PerkinElmer (Waltham, MA, USA). Anti-CLIC3 antibody (ab128941, catalog number; EPR8243), Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 568-conjugated anti-mouse IgG antibodies were from Abcam (Cambridge, UK). Anti-β-actin antibody (8H10D10, catalog number; 3700S) was from Cell Signaling Technology (Beverly, MA, USA). Horse-radish peroxidase-conjugated anti-rabbit and anti-mouse IgGs were from Millipore (Bedford, MA, USA). 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was from Research Biochemicals International (Natick, MA, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was from Dojindo Laboratories (Kumamoto, Japan). Polyethylenimine Max (PEI-Max) and Differential Quik Stain kit were from Polysciences Inc. (Warrington, PA, USA). All other reagents were of molecular biological grade or of the highest grade of purity available.
8
Colony Forming Assay Protocol
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Approximately 3,000 cells were added to each well of a six-well culture plate, and each experiment was performed in triplicate. After 12 days of culture at 37°C, the cells were fixed and stained using a Differential Quik Stain Kit (Polysciences, Inc., Warrington, PA, USA). Visible colonies were manually counted under a microscope.
9
Chemotaxis Assay for SMCs
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The primary culture of SMCs was cultured on a matrigel-coated chamber with 8-μm pore (Corning, One Riverfront Plaza Corning, NY). 100 ng/ml recombinant human PGDF-BB protein (R&D Systems, Minneapolis, MN) or 100 ng/ml recombinant human AREG protein (R&D Systems) was added in a lower chamber and migrated SMCs across pores within 24 h were counted after staining by a Differential Quik Stain Kit (Polysciences Inc., Warrington, PA).
10
Modified Giemsa Cell Staining
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Modified Giemsa staining was achieved by using Differential Quik Stain Kit, following the manufacturer’s instructions (Polysciences). Cell staining images were acquired on Olympus BX43 microscope with a camera (DP72) using cellSens Standard software (version 1.4.1; Olympus).
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