The effect of AR down-modulation on E006AA cell migration and invasion was performed using 8-µm transwell filters (Corning) with minor modification as described previously 21 (link). For the invasion assay, the upper compartment was coated with 50 µg Matrigel (BD Biosciences) to form a matrix barrier. A suspension of cells (4 × 104 in 200 µl) in basal medium containing 0.1% BSA was added to the upper compartment. The lower compartment was filled with 400 µl basal medium containing 1% FBS as a chemoattractant. After 24 h, the non-migratory cells on the upper surface were removed by a cotton swab and the cells on the lower surface were fixed and stained with the Diff-Quick solution (Dade Behring, Deerfield, Illinois). Migrated or invaded cells in each transwell filter were counted in ten randomly selected fields (at 40X magnification). The experiment was performed in quadruplicates and repeated at least three times independently.
Diff quick solution
Manufactured by Siemens
Sourced in United States
The Diff-Quick solution is a staining kit used in clinical laboratories for the rapid differential staining of blood smears. The solution consists of three stains that are applied sequentially to fix, stain, and differentiate the cellular components of the blood sample. This process allows for the identification and enumeration of different types of blood cells, which is an essential tool in hematology and diagnostic pathology.
Automatically generated - may contain errors
6 protocols using diff quick solution
1
Transwell Assay for Cell Migration and Invasion
2
Bronchoalveolar Lavage Cell Counting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bronchoalveolar lavage was performed and total cell numbers in lavage fluid were determined using a Coulter Counter (IG Instrumenten-Gesellschaft AG, Basel, Switzerland). Differential cell counts were performed on cytospins stained with Diff-Quick solution (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, IL, USA). Percentages of eosinophils and neutrophils were determined within a total count of 200 cells per sample.
3
Transwell Assay for Cell Migration and Invasion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effect of AR silencing on cell migration was performed using 8-μm transwell filters (Costar, Corning, NY) with modification as described previously 39 (link). For the invasion assay, the upper compartment was coated with 50 μg Matrigel (BD Biosciences, San Jose, CA) to form a matrix barrier. A suspension of cells (20 × 104 per filter for migration and 40 x 104 per filter for invasion) in medium containing 1% FBS and 0.1% BSA was added to the upper compartment. The lower compartment was filled with 400 μl basal medium containing 10% FBS as chemoattractant. After 24 h for migration or 48 h for invasion, the non-migratory cells on the upper surface were removed by a cotton swab and the cells on the lower surface were fixed and stained with the Diff-Quick solution (Dade Behring, Deerfield, Illinois). Migrated or invaded cells in each transwell filter were counted from ten randomly chosen fields. The experiment was performed in quadruplicate and repeated three times independently.
4
Bronchoalveolar Lavage for Cellular Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were sacrificed by intraperitoneal (i.p.) administration of 90 mg/kg body weight sodium pentobarbital. Bronchoalveolar lavage fluid (BALF) was obtained by lavaging the lungs with 3 mM EDTA in PBS [32 (link)] and the cellular composition was determined by hemocytometer cell counts and differential counts of cytospins after staining with Quick-Diff solution (Siemens; Medical Solutions Diagnostics, Tarrytown, NY, USA).
5
Quantifying Inflammatory Mediators in Mouse BAL
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were sacrificed by intraperitoneal (i.p.) administration of 90 mg/kg of body weight sodium pentobarbital. Bronchoalveolar lavage (BAL) was performed by washing the lungs with 3 mM EDTA in PBS (16 (link)) and cellular composition was determined by hemocytometer cell counts and differential counts of cytospins after staining with Quick-Diff solution (Siemens; Medical Solutions Diagnostics, Tarrytown, NY). Cell-free BAL fluid (BALF) was used to determine levels of IL-13 (4–500 pg/ml) and IFN-α (31.3–20.00 pg/ml) using ELISA kits (Ready-SET-Go; eBioscience, San Diego, CA), and levels of IL-1α (Minimal Detectable Concentration (MDC); 1.3 pg/mL), IFNγ (MDC; 0.8 pg/mL), TNFα (MDC; 1.9 pg/mL), IL-1β (MDC; 2.8 pg/mL), IL-10 (MDC; 2.1 pg/mL), IL-6 (MDC; 0.9 pg/mL), IFN-β (MDC; 4 pg/mL) using the LEGENDplex mouse inflammation panel (BioLegend, San Diego, CA). LEGENDplex panel was acquired on LSRII running FACS-Diva software (both obtained from BD Bioscience) and analyzed using BioLegend data analysis software. Results are from ≥4 mice per group (biological replicates) and 2 technical replicates per mouse.
6
Bronchoalveolar Lavage Cytokine Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were sacrificed by intraperitoneal (i.p.) administration of 90 mg/kg of body weight sodium pentobarbital. Bronchoalveolar lavage fluid (BALF) was obtained by lavaging the lungs with 3 mM EDTA in PBS (11 (link)), and cellular composition was determined by hemocytometer cell counts and differential counts of cytospins after staining with Quick-Diff solution (Siemens; Medical Solutions Diagnostics, Tarrytown, NY). Cell-free BALF was used to determine levels of IL-13 (4 to 500 pg/ml), IFN-β (1.9 to 500 pg/ml), and IFN-γ (15.6 to 1,000 pg/ml) using enzyme-linked immunosorbent assay (ELISA) kits (Ready-Set-Go; eBioscience, San Diego, CA, or BioLegend). Results are from 5 mice per group (biological replicates) and 3 technical replicates per mouse.
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!