Human lung carcinoma cell lines A549 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were incubated in Kaighn’s modification of F-12 Ham Nutrient Mixture supplemented with 10% fetal bovine serum (FBS) and antibiotics (1% Antibiotic-Antimycotic 100 × and 50 × 10−3 g L−1 gentamicin; Biosera) at 37 °C, 95% humidity and 5% CO2. The cells (10,000/cm−2) were seeded on 12-well μ-Chamber slides (ibidi GmbH, Martinsried, Germany) on 6, 12 and 96-well plates (TPP, Trasadingen, Switzerland) and left to settle for 24 h. This incubation method has been published previously [39 (link)].
μ chamber slides
Manufactured by Ibidi
Sourced in Germany
μ-Chamber slides are a type of lab equipment designed for cell culture applications. They provide a controlled environment for studying cell behavior and interactions. The slides feature a built-in chamber that allows for precise regulation of the cell culture conditions, such as temperature, humidity, and gas exchange.
Automatically generated - may contain errors
Lab products found in correlation
Antibiotic antimycotic 100, by Biosera (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
L 1 gentamicin, by Biosera (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Gentamicin, by Biosera (1 mentions)
Mem medium, by PAN Biotech (1 mentions)
Ccd 18co, by PAN Biotech (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Ccd 18co, by American Type Culture Collection (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
4 protocols using μ chamber slides
1
Culturing Human Lung Carcinoma Cells
2
Glycogen Immunofluorescence Staining Protocol
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Cells grown in μ chamber slides (Ibidi) were PBS washed and fixed with 4% PFA for an hour at RT. After PBS wash, cells were permeabilised and blocked in 10% horse serum and 0.1% triton in PBS. They were then incubated overnight at 4 °C in primary antibody solution (1:200 glycogen, gift of Otto Baba) in 10% horse serum in PBS. After three washes with PBS, cells were incubated in the secondary solution in PBS (1:1000, Alexa Fluor 555 goat anti-mouse). Slides were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Labs) and imaged using Zeiss 880 LSM confocal microscope.
3
Biscoumarin Effects on A549 and CCD-18Co Cells
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Human lung carcinoma cell line A549 and CCD-18Co colon fibroblasts were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The A549 cells were cultured in a complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), and CCD-18Co cells were cultured in an MEM medium (PAN-Biotech GmbH, Aidenbach, Germany). The media were supplemented with 10% fetal bovine serum (FBS; Biosera, Nuaille, France) and antibiotics (1% Antibiotic-Antimycotic 100× and 50 × 10−3 g l−1 gentamicin; Biosera) at 37 °C, 95% humidity, and 5% CO2.
Prior to the selected treatments, cells were seeded on 12-well μ-Chamber slides (ibidi GmbH, Martinsried, Germany) and 6 and/or 96-well plates (TPP, Trasadingen, Switzerland) and left to settle for 24 h. The biscoumarin derivative solutions (at concentrations ranging from 10–100 µM) were then added to cells for 24 or 48 h, and analysis was subsequently performed.
Prior to the selected treatments, cells were seeded on 12-well μ-Chamber slides (ibidi GmbH, Martinsried, Germany) and 6 and/or 96-well plates (TPP, Trasadingen, Switzerland) and left to settle for 24 h. The biscoumarin derivative solutions (at concentrations ranging from 10–100 µM) were then added to cells for 24 or 48 h, and analysis was subsequently performed.
4
Imaging Effector-Target Cell Interactions
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For experiments with adherent target cells, cells were harvested using 0.05% EDTA cell detachment solution to avoid ligand degradation by trypsin. Target cells were incubated on cell culture-treated eight-well μChamber slides (Ibidi) for 4–6 h at 37°C, 5% CO2. Effector cells were added at a 2:1 effector:target ratio and co-incubated for an additional 20 min. Cells were rinsed and fixed using 1.6% v/v para-formaldehyde. Samples were blocked using 3% w/v bovine serum albumin for 30 min and incubated with 10 μg/mL biotinylated monoclonal antibody or isotype control biotinylated antibody in PBS containing 0.05% v/v Triton X-100. Samples were washed thoroughly and incubated with streptavidin Alexa Fluor 647 for 30 min at RT. For F-actin labeling, samples were incubated in PBS solution containing 10 units/mL Rhodamine Phalloidin (Life Technologies), and 0.05% v/v Triton X-100. Nuclei were stained with 2 mg/mL Hoechst 33342 (Sigma) and ProLong Gold antifade (Life Technologies) reagent was added to the samples before mounting. Images were acquired using LSM510 Meta (Zeiss) laser scanning confocal microscope equipped with a 63× (NA 1.4) DIC oil objective.
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