Lungs were lavaged with 1 mL PBS and 20 μl of total bronchoalveolar lavage fluid (BALF) was diluted in 10 mL Isoton II diluent and counted on a Z2 particle count and size analyzer (Beckman Coulter, Fullerton, CA).
Z2 particle count and size analyzer
Manufactured by Beckman Coulter
The Z2 Particle Count and Size Analyzer is a laboratory instrument designed to accurately measure the size and count of particles in a sample. It utilizes the principle of electrical impedance to detect and analyze the particles suspended in a liquid or gas medium. The core function of the Z2 Particle Count and Size Analyzer is to provide precise data on the particle size distribution and concentration within a given sample.
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5 protocols using z2 particle count and size analyzer
1
Bronchoalveolar Lavage Cell Counting
2
Lin37 Mutant Cell Volume Analysis
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Proliferating and confluent Lin37-KO cells (clone 632–2) were treated with doxycycline (500 ng/ml) to express wild-type Lin37, the CD1+2 MuvB binding mutant or luciferase together with GFP from pRTS episomes. Confluence was maintained over 48 hr. GFP-positive cells were isolated with a BD FACSAria II cell sorter prior to cell volume analysis. Cell volumes were assessed by Coulter counter analysis (Beckman-Coulter Z2 Particle Count and Size Analyzer). For each sample, three technical replicates with approximately 30,000 cells were examined.
3
Colony Formation Assay with Dexamethasone
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Cells were counted using a Beckman Coulter Z2 Particle Count and Size Analyzer and plated at a density of 400 cells per well of a 6-well plate. Cells were treated the next day with serial dilutions of dexamethasone. Plates were kept in the cell culture incubator until termination of assay. After 2-3 weeks, colonies were stained with crystal violet staining solution (0.5% crystal violet, 3% formaldehyde solution), rinsed in water and imaged.
4
Fibroblast Proliferation and Apoptosis Assay
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Fibroblasts isolated from 4-OHT-treated WT or TERT CKO lungs at passage 1 were seeded into 96-well plates (5×103 cells/well). After 24 hours, 10 ng/ml of PDGF (R & D systems) was added and cultured for the indicated times up to 96 hours. The cell number was counted using a Z2 Particle Count and Size Analyzer (Beckman Coulter, Inc., Indianapolis, IN). Where indicated, 10 μl of WST-1 reagent (Roche) was added 48 hours after PDGF treatment for measuring cell proliferation by WST-1 assay as described before [25 (link)]. Apoptosis assay was performed in WT or TERT KO MLF treated with 5 ng/ml TNFα and 500 ng/ml CHX using TACS AnnexinV-FITC kit (R & D systems) and propidium iodide (PI) as described before [25 (link)]. Analysis was undertaken using a FACS Caliber flow cytometer (BD Biosciences). Apoptotic cells were identified as an annexin V+/PI- population.
5
Generating Stable Cell Lines Expressing hTERT Mutants and FOXO4
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Amphotropic retroviruses were created using the retroviral vectors pBABE-puro, pBABE-puro-hTERT T249A or pBABE-puro-hTERT T249E for making stable cell lines expressing hTERT mutants. After infection, polyclonal cell populations were purified by selection with puromycin (2 µg/ml) for 3 days70 (link).
We used the following short-hairpin RNA (shRNA) vectors, shFOXO4-1 (TRCN0000010291) and shFOXO4-2 (TRCN0000039720), constructed by The RNAi Consortium and the sequences are listed in Supplementary Table7 . shGFP was used as the control. These vectors were used to make amphotropic lentiviruses, and the cell populations were selected with puromycin (2 µg/ml) for 3 days.
For generating stable cell lines expressing FOXO4, we constructed the retroviral vector pBABE-hygro (pBh)-FOXO4. After infection of pBABE-hygro or pBh-FOXO4, the cells were selected with hygromycin B (200 µg/ml) for 7 days.
To generate proliferation curves, cells were plated in triplicate and counted in a Z2 Particle Count and Size Analyzer (Beckman-Coulter).
We used the following short-hairpin RNA (shRNA) vectors, shFOXO4-1 (TRCN0000010291) and shFOXO4-2 (TRCN0000039720), constructed by The RNAi Consortium and the sequences are listed in Supplementary Table
For generating stable cell lines expressing FOXO4, we constructed the retroviral vector pBABE-hygro (pBh)-FOXO4. After infection of pBABE-hygro or pBh-FOXO4, the cells were selected with hygromycin B (200 µg/ml) for 7 days.
To generate proliferation curves, cells were plated in triplicate and counted in a Z2 Particle Count and Size Analyzer (Beckman-Coulter).
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