BALF was collected by intratracheal intubation and 5 serial lung lavages using 1 ml 0.9% saline. Immune cells were isolated from BALF by centrifugation at 230 g for 10 minutes, and pellets were resuspended in 100 μl of 0.9% saline for total and differential cell analyses. Cell-free BALF was used for measurement of surfactant lipids and total protein. Lipids were extracted from cell-free BALF by the Bligh and Dyer method50 (link). Saturated phosphatidylcholine was isolated using the osmium tetroxide based method of Mason et al.51 (link) and quantitated by phosphorous measurement. Cytospin slides were prepared at a cell density of 1 × 106 cells/ml and stained with the Shandon Kwik-Diff stain kit (Thermo Fisher Scientific). A total of 300 cells was counted manually per slide to determine the percentage of monocyte/macrophages, lymphocytes and neutrophils.
Shandon kwik diff stain kit
Manufactured by Thermo Fisher Scientific
The Shandon Kwik-Diff stain kit is a simple and rapid staining solution for the differential staining of blood smears and other cytological samples. The kit provides a three-step staining process that produces a Romanowsky-type stain, enabling the visualization of cellular morphology.
Automatically generated - may contain errors
Lab products found in correlation
Wst 1 viability assay, by Roche (1 mentions)
Matrigel, by BD (1 mentions)
Methocult h4434 medium, by STEMCELL (1 mentions)
Aperio imagescope software, by Leica (1 mentions)
Matrigel matrix, by Corning (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Lonza (1 mentions)
Hanks balanced salt solution (hbss), by Corning (1 mentions)
5 protocols using shandon kwik diff stain kit
1
Characterization of Immune Cells in BALF
2
Evaluating the Impact of ML246 on Ovarian Cancer Cell Viability and Invasion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SKOV3 and OVCAR3 cells were treated with vehicle or ML246 for 72 h at the indicated concentrations. Cell viability was measured using the WST-1 viability assay as per the manufacturer’s instructions (Roche, Switzerland).
In vitro invasion assays were performed on SKOV3 and OVCAR3 using 24-well transwell units with polycarbonate filters (pore size: 8 μm) coated on the upper side with reconstituted basement membrane matrix, Matrigel (BD Biosciences, USA). 5 × 105 cells were added to the transwell in serum free media. Media with FBS was added to the outer compartment as the chemoattractant. Cells were treated with DMSO or ML246, and cultured for 72 h at 37 °C. Cells remaining on the upper side of the transwell were scraped off with a cotton swab. Cells remaining on the underside of the membrane were fixed and stained using the commercially available, Shandon Kwik-Diff stain kit (ThermoFisher Scientific). Cells were counted in four fields and the mean+ SEM was calculated. This experiment was performed in triplicate in three independent experiments.
In vitro invasion assays were performed on SKOV3 and OVCAR3 using 24-well transwell units with polycarbonate filters (pore size: 8 μm) coated on the upper side with reconstituted basement membrane matrix, Matrigel (BD Biosciences, USA). 5 × 105 cells were added to the transwell in serum free media. Media with FBS was added to the outer compartment as the chemoattractant. Cells were treated with DMSO or ML246, and cultured for 72 h at 37 °C. Cells remaining on the upper side of the transwell were scraped off with a cotton swab. Cells remaining on the underside of the membrane were fixed and stained using the commercially available, Shandon Kwik-Diff stain kit (ThermoFisher Scientific). Cells were counted in four fields and the mean
3
Clonogenic Assay for Hematopoietic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Clonogenic colony-forming capacities of healthy BM and mobilized PB (10,000–30,000 cells), AML BM mononuclear cells (10,000–30,000 cells), and total dissociated EB cell suspensions (20,000–30,000 cells) plated in Methocult H4434 medium (Stem Cell Technologies, Vancouver, Canada, http://www.stemcell.com ) were monitored between days 7 and 16, and colonies were quantified based on morphology between 14 and 16 days. Individual colonies were isolated and assessed for single-cell morphology, and full wells were collected for fluorescence in situ hybridization (FISH) analysis. Depending on number of colonies generated in CFU assay, single-cell morphologies of at least three colonies were analyzed to confirm colony quantification criteria and evaluate the maturity of colonies. Briefly, colonies were isolated and resuspended in 100 µl PBS and spun onto microscope slides using the Shandon Cytospin 3 (Block Scientific, Inc., Bellport, NY, http://www.blockscientific.com ). Morphological features were visualized by Giemsa-Wright staining performed using Shandon Kwik-Diff Stain Kit (Thermo Scientific, Waltham, MA, http://www.thermoscientific.com ). Images were acquired using ScanScope CS digital slide scanner with Aperio ImageScope software (Leica Biosystems).
4
Cell Migration and Invasion Assays
5
Lung Cell Preparation for Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lungs were instilled with 3 ml Hank's balanced salt solution (HBSS; Corning) containing 0.05 mM EDTA (Lonza) and cell suspensions (≈10,000 cells in 100 μl) were centrifuged at 800 rpm for 5 min in a Shandon CytoSpin 3 cytocentrifuge (Cell Preparation System). The cytospin pellets were air dried on glass slides, stained with Shandon Kwik–Diff™ Stain kit (Thermo Scientific) and were mounted with Richard–Allan Scientific™ Cytoseal™ 60 (Thermo Scientific).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!