HES was purchased from Braun Medical Inc. (US). Fura-2- acetoxymethyl ester (Fura-2AM), CM-H2DCFDA, calcein-AM, the 7AAD viability staining solution and the pHRodo Red Zymosan A Bioparticles kit were obtained from Thermo Fisher (ON, Canada); dextran T500, cytochrome C, Hemacolor ® Rapid staining kit from Sigma-Aldrich (ON, Canada); lymphocyte separation medium, RPMI-1640, BSA (bovin serum albumin) and FBS (fetal bovine serum) from Wisent Bioproducts (QC, Canada); and the ChemoTx microplates (101–8) from NeuroProbe (MD, USA).
Hemacolor rapid staining kit
Manufactured by Merck Group
Sourced in Germany, United States
The Hemacolor rapid staining kit is a laboratory product manufactured by Merck Group. It is used for the rapid staining of blood smears and other cytological preparations. The kit provides a reliable and efficient way to stain cells for microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Easysep human eosinophil isolation kit, by STEMCELL (2 mentions)
Pancoll human, by PAN Biotech (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Dextran t500, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Wisent (1 mentions)
7 aad viability staining solution, by Thermo Fisher Scientific (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
Rpmi 1640, by Wisent (1 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Bromocresol green albumin assay kit, by Merck Group (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Chemotaxis chamber, by Neuro Probe (1 mentions)
A light microscope, by Zeiss (1 mentions)
Sigmastat 3, by Grafiti LLC (1 mentions)
Bx61 upright microscope, by Olympus (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
19 protocols using hemacolor rapid staining kit
1
Immune Cell Functional Assays
2
Characterization of Lung Injury in hTgβc Mice
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On day 12, hTgβc mice were euthanized, blood collected and BAL performed as previously described.31 , 32 Cytospin slides stained with a Hemacolor® Rapid Staining Kit (Sigma‐Aldrich, USA) were used for differential cell counting. The left lung was fixed in 10% neutral‐buffered formalin for histology, the right superior lobe was excised for flow cytometry and the remaining lobes was snap‐frozen in liquid nitrogen and stored at −80°C as previously described.31 , 32 Lung injury was blindly scored as previously described,33 where the degree of inflammatory cell infiltration, epithelial/endothelial destruction and alveolar septal thickening was used to generate an aggregate score. Lactate dehydrogenase (LDH) levels in the BAL fluid (BALF) were measured using the Pierce™ LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, USA). Lung oedema was determined by measuring serum albumin levels in the BALF using the Bromocresol Green Albumin Assay kit (Sigma‐Aldrich, USA). Gelatinase activity in the BALF was measured using the EnzChek™ Gelatinase Kit (Thermo Fisher Scientific, USA). Cell‐free double‐stranded DNA (dsDNA) in the BALF was determined using the Quant‐iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, USA). Total RNA and real‐time quantitative PCR (RTqPCR) were then performed as previously described.33 , 34
3
BAL Cell Differential and VOC Analysis
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Total viable cells collected from BAL were calculated using a haemocytometer. Cytospin was then performed, and the slides were stained using the Hemacolor® Rapid Staining Kit (Sigma-Aldrich, US) for differential cell counting [20 (link), 21 (link)]. The remaining fluid was centrifuged, and the supernatant (cell-free BAL fluid) was collected and stored at -80 °C for volatile organic compound (VOC) analysis.
4
Cell Invasion Assay Protocol
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Cell invasion was assessed using a chemotaxis chamber (Neuro Probe, Gaithersburg, MD, USA). The cells (5×104 cells in 0.35-ml serum-free DMEM per well) were seeded into the top chamber of a 10-well invasion chamber assay plate. DMEM supplemented with 10% FBS was placed in the lower chamber, and a matrigel-coated membrane was inserted between the two chambers. After incubation for 24 h at 37°C, the membranes were fixed and stained with a Hemacolor rapid staining kit (Merck KGaA). Cells from five random microscopic fields (each 0.5 mm2) were counted using a hematocytometer under a light microscope (Zeiss).
5
Chemotactic Migration of Porcine MSCs
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The migratory effect of recombinant human CCL25 (Order No 300-45; PeproTech) on porcine MSC (n = 3 donors) was assessed using 96-well ChemoTx plates with 8 µm pore-size membranes (Neuro Probe, Gaithersburg, MD, USA) [15 (link)]. Lower wells contained 40 µL deprivation medium (DMEM, 0.1% FBS) plus different CCL25 concentrations (0, 0.1, 1, 10, 100, 250, 500, 1000 nmol/L). Deprivation medium with 10% FBS served as the positive control. Upper wells were supplied with 3 × 104 MSC per 37.5 µL deprivation medium and incubated for 20 h. Migration assay was performed in triplicates. Migrated cells were fixed with methanol and stained using the Hemacolor Rapid Staining Kit (Merck). Photographs were taken and cell amount per field was enumerated with the Cell Counter plugin of ImageJ software (NIH, Bethesda, MD, USA). Statistical analysis was performed with SigmaStat 3.5 (Systat Software, Erkrath, Germany). Normal distribution of data was checked and one-way analysis of variance (ANOVA) for correlated samples was used with post hoc analysis according to Fishers Least Significant Difference-Test.
6
Cytospin Cell Preparation and Staining
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Sorted cells were mounted on Superfrost PLUS slides (Thermo Scientific) using a Cytospin centrifuge (Cytospin 3, Shandon) for 5 min at 500 rpm. Cells were fixed with 2% paraformaldehyde for 10 min at room temperature and stained with the Hemacolor Rapid Staining Kit (Merck Millipore). Images were collected on BX61 upright microscope (Olympus) using ×100 objective with immersion oil and captured with a CCD camera. Images were then analyzed and processed with ImageJ (NIH) and Adobe Photoshop 5.5 (Adobe).
7
Murine Bronchoalveolar Lavage Analysis
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After the mouse was euthanized, the trachea was intubated with a 20-gauge catheter (Terumo). Murine lungs were lavaged with 0.7-ml ice cold PBS 3 times (a total of 2.1 ml), and the BALF was collected. After centrifugation at 500 × g for 5 min at 4 °C, the supernatant was aliquoted and stored at −80 °C; BALF protein concentration was measured using BCA protein assay kit (Thermo Fisher). Cell pellets were re-suspended in 100 μL PBS. The cell number was quantified using 10 μL cell suspension by a Countess II Automated Cell Counter (Thermo Fisher), and cytospin slides were prepared using 40 μL of the cell suspension with 160 μL of PBS [28.23 × g (500 r.p.m.) for 5 min]. Slides were stained using the Hemacolor Rapid staining kit (EMD Millipore), and the numbers of macrophages, leukocytes and neutrophils were counted in a total of at least 200 cells.
8
Transwell Invasion Assay for ANO1
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Cell invasion activity was evaluated in transwell 24-insert plate chambers (8-µm pore size Corning Costar Transwell Permeable Supports; Fisher Scientific, Waltham, MA, USA) coated with BioCoat Matrigel (BD Matrigel Basement Membrane Matrix; diluted 1:50 in RPMI-1640 serum-free medium). After transfection with ANO1 siRNA or negative control siRNA for 24 h, 3 × 105 FRO cells/mL were plated in upper chambers in 100 μL of serum-free RPMI medium. The lower chambers were filled with RPMI medium supplemented with 10% FBS. The transwells were incubated for 24 h to allow for cell migration. Following incubation, cells from the upper side of the insert filter were completely removed using a cotton swab and cells that invaded through the collagen-coated membrane were fixed in methanol and stained with a Hemacolor rapid staining kit (Merck Millipore, Germany). For quantification, cells were counted under a microscope (Axiocam HRc; Zeiss, Jena, Germany) in six random fields at 20× magnification. These experiments were performed in triplicate.
9
Cytospin-based Visualization of TOM+ and TOM- Dendritic Cells
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TOM+ cDC2 and TOM− DC2 were sorted from CD11c+ enriched splenocytes from 11-day-old Clec9acre/creRosaTOM mice as live, single, autofluorescence-negative, CD11c+MHCII+CD11b+ TOM+ or TOM− cells. Cells (2 × 104) were spun onto a microscope slide in a Shandon Cytospin 2 for 5 min at 8000 r.p.m. and stained with Hemacolor® rapid staining kit (Merck). Microscopy was performed at the Core Facility Bioimaging of the Biomedical Center using a Leica DM 2500 LED microscope with a ×100 magnification.
10
Transwell Migration and Invasion Assays
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Transwell migration and invasion assays (Corning Life Science) were performed using 24-well plates with polycarbonate membrane inserts with an 8 μm pore size. Briefly, the cells supplemented with serum-free medium were seeded into the Matrigel-coated chambers (for invasion) or Type I collagen-coated chambers (for migration). The lower chambers contained RPMI1640 with 10% FBS. After incubation for 24 h, the migrated or invaded cells were fixed and stained using the Hemacolor® rapid staining kit (Merck, Darmstadt, Germany). The cells at the bottom of the membrane were counted under a light microscope.
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