The human lung carcinoma epithelial cell line A549 (ATCC, CCL-185) was maintained by serial passage in Dulbecco’s modified Eagle medium (DMEM) (Ref. code: 11995–065, Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). Fungal infections were performed as described [64 (link)]. In short, cells were seeded at a concentration of 2 105 cells mL-1 and cultured at 37°C with 5% CO2 until a confluent monolayer was formed. Cells in 12, 24 or 48 wells plates (Corning®, Costar®) were challenged with 2 106 conidia mL-1 DMEM + 10% FBS, resulting in a multiplicity of infection (MOI) of 1. Cells were cultured in 48 wells plates containing 8 mm glass coverslips (ThermoFisher Scientific) for internalization, association and germination experiments, in 12 wells plates for the IL-8 secretion or in 24 wells plates containing 12 mm glass coverslips (VWR International) for the identification of apoptotic and necrotic cells (see below).
12 mm glass coverslips
Manufactured by Avantor
12 mm glass coverslips are thin, transparent glass discs used to cover and protect specimens in microscopy applications. They provide a flat, uniform surface for mounting and viewing samples under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
8 mm glass coverslips, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Prolong antifade kit, by Thermo Fisher Scientific (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Pclamp 10, by Molecular Devices (1 mentions)
Digidata 1440a, by Molecular Devices (1 mentions)
Axopatch 200b amplifier, by Molecular Devices (1 mentions)
Fugene 6, by Promega (1 mentions)
Rsc 200, by Bio-Logic (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
24 well plate, by Avantor (1 mentions)
6 well plate, by Avantor (1 mentions)
Laminin, by Roche (1 mentions)
6 protocols using 12 mm glass coverslips
1
A549 Cell Line Fungal Infection Assay
2
LXA4 Modulation of LPS-Induced Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study the effect of LXA4 on LPS‐induced inflammation, cells were stained as previously reported (Diomede et al., 2017 ). For monoculture, PDLCs (2 × 103 cells/well) were seeded in 8‐chambered glass slides (Millicell). In coculture, PDLCs were cultured in a 24‐well plate with 12‐mm glass coverslips (Deckgläser, VWR International B.V.). At day 1, the cells were challenged with 10 μg/ml of E. Coli LPS for 24 hr in a 37°C incubator with 5% CO2. At day 2, the cells were washed twice with PBS and then treated with 100 ng/ml LXA4 for an additional 24 hr under the same storage conditions. At day 3, the cells were fixed for 10 min at RT with 4% paraformaldehyde in PBS, pH 7.2, followed by blocking with 1% BSA. Cells were then stained with primary monoclonal antibodies anti‐human TLR4, MyD88, NF‐κB, followed by GTα (AFS94) as secondary antibody under the conditions outlined in Table 1 . Before mounting for microscopic observation, samples were briefly washed in distilled water and cell nuclei were stained with 4‐6‐diamidino‐2‐phenylindole (DAPI) (1:1,000) for 10 min at 37°C (Invitrogen).
3
Neutrophil Response to Pseudomonas aeruginosa
4
Apoptosis and Necrosis Assay in A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells were grown on 12-mm glass coverslips (VWR international) until a confluent cell layer was formed. The A549 cells were challenged with conidia for 2 h, after which unbound conidia were removed by washing three times with pre-warmed DMEM + 10% FBS. Exposure of the A549 cells to the conidia continued for another 2 or 10 h and washed twice with PBS. Staining of the apoptotic and necrotic cells was based on [65 (link)]. In short, 100 μg mL-1 acridine orange (AO) (VWR International) and 100 μg mL-1 ethidium bromide (EB) (Sigma Aldrich) was added to PBS for each experiment and added to the cells. The cells were immediately mounted on a coverslip with FluorSavetm and examined by fluorescence microscopy within 15 min after adding the dye. The experiments were performed using biological triplicates with pictures taken at 10 random places on the slide. At least 100 cells per strain were analysed per replicate.
5
Whole-Cell Patch-Clamp Recordings of Panx1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells were plated onto 12 mm glass coverslips (VWR) in wells of a six-well plate and transfected 24 hr later with 500–800 ng plasmid DNA using FUGENE 6 (Promega) according to the manufacturer’s instructions. Recordings were performed ~16–24 hr later using borosilicate glass micropipettes (Harvard Apparatus) pulled and polished to a final resistance of 2–5 MΩ. Pipettes were backfilled with (in mM) 147 NaCl, 10 EGTA, 10 HEPES pH 7.0 with NaOH. Patches were obtained in external buffer composed of (in mM) 147 NaCl, 10 HEPES pH 7.3 with NaOH, 13 glucose, 2 KCl, 2 CaCl2, 1 MgCl2. A rapid solution exchange system (RSC-200; Bio-Logic) was used to perfuse cells with CBX or various salt solutions. Currents were recorded using an Axopatch 200B amplifier (Axon Instruments), filtered at 2 kHz (Frequency Devices), digitized with a Digidata 1440A (Axon Instruments) with a sampling frequency of 10 kHz, and analyzed with the pClamp 10.5 software (Axon Instruments). For voltage step recordings, Panx1-expressing cells were held at −60 mV and stepped to various voltage potentials for 1 s in 20 mV increments before returning to −60 mV. For ramp recordings, cells were held at −60 mV, and ramped between −100 mV and + 100 mV over 3 s duration.
6
Primary Hippocampal Neuron Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary hippocampal neurons from Wistar rat pups at P0 were cultured in 6-well plates (VWR) or in 24-well plates (VWR) on 12 mm glass coverslips (VWR) coated with poly-D-lysine (Sigma Aldrich) and laminin (Roche). After tissue homogenization, cells were counted in a B€ urker chamber and seeded at the density of $300,000 cells or $75,000 cells per well, respectively. After 1.5-2 h, plating medium (MEM supplemented with 10% fetal bovine serum, 0.45% glucose, 2 mM glutamine and penicillin/streptomycin 100 U/ml-100 mg/mL) was replaced with defined maintenance medium (neurobasal supplemented with B27, 2 mM glutamine and penicillin/streptomycin 100 U/ml-100 mg/mL).
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!