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Lab tek chambered coverglass

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The Lab-Tek chambered coverglass is a multi-well cell culture system designed for microscopic examination. It provides a sterile environment for growing and observing cells. The coverglass is made of high-quality borosilicate glass and is available in various well configurations to suit different experimental needs.

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Lab products found in correlation

Lsm 780 confocal microscope, by Zeiss (6 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Lsm 510 meta, by Zeiss (4 mentions) Sp8 microscope, by Leica camera (4 mentions) Lsm 510 meta confocal microscope, by Zeiss (4 mentions) Lsm 710, by Zeiss (4 mentions) Poly l lysine (pll), by Merck Group (3 mentions) Tcs sp5, by Leica camera (3 mentions) Axio observer z1, by Zeiss (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Ti e perfect focus microscope, by Nikon (2 mentions) 6 well chambers, by BD (2 mentions) Trypsin edta, by Thermo Fisher Scientific (2 mentions) Lsm 710 confocal laser scanning microscope, by Zeiss (2 mentions) Tcs sp5 confocal laser scanning microscope, by Leica Microsystems (2 mentions) Csu x1, by Yokogawa (2 mentions) Mounting solution, by Thermo Fisher Scientific (2 mentions) Lsm 510, by Zeiss (2 mentions) Confocal laser scanning microscope, by Zeiss (2 mentions) Turbofect, by Thermo Fisher Scientific (2 mentions)

94 protocols using lab tek chambered coverglass

1

Cell Viability and Proteasomal Activity Assays

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Lab-Tek chambered cover glass was from Nunc (Rochester, NY, USA). Other tissue culture supplies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Living cell numbers were quantitated by ATP assay using the CellTiter-Glo Luminescent Cell Viability Assay Kit (catalog# G755A, Promega, Madison, WI, USA), as previously reported (Hamano et al., 2012 (link), 2016 (link); Shirafuji et al., 2018 ). The ROCK inhibitor H1152 was from Calbiochem (catalog #555552–500UG, Darmstadt, Germany) and Y-27632 was from WAKO (catalog #257–00511, Tokyo, Japan). Suc-LLVY-AMC fluorogenic substrate (Biomol International) for 20S chymotrypsin-like activity and Bz-Val-Gly-Arg-AMC (Biomol International) fluorogenic substrate for 26S proteasomal activity assay were from Biomol International (Plymouth Meeting, PA, USA). All other chemicals were from Sigma (Saint Louis, MO, USA) unless stated otherwise.
Hamano T., Shirafuji N., Yen S.H., Yoshida H., Kanaan N.M., Hayashi K., Ikawa M., Yamamura O., Fujita Y., Kuriyama M, & Nakamoto Y. (2019). Rho-kinase ROCK inhibitors reduce oligomeric tau protein. Neurobiology of aging, 89, 41-54.
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2

Imaging Cells on Collagen-Coated Surfaces

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Cells were grown on collagen-coated coverslips or chambered slides (Nunc Labtek Chambered Coverglass) for 48 h after transfection if not stated otherwise. For analysis of fixed cells, samples were treated with 4% paraformaldehyde (30 min, room temperature) or methanol (10 min, −20°C), permeabilized with 0.1% Triton X-100 (15 min, room temperature), and mounted in Mowiol. Live cells were imaged in medium supplemented with 25 mM HEPES at 37°C. Confocal imaging used a Zeiss LSM780 confocal microscope and a Plan-Apochromat 63×/1.4 oil immersion objective. Confocal videos were recorded at different speeds. TIRF imaging was performed with an Olympus IX71 TIRF Microscope customized to include a heated incubation chamber, an objective-type TIRF microscopy setup from TILL Photonics, a monochromator for epifluorescence excitation, and a controller allowing hardware-controlled fast switching between total internal reflection fluorescence and epifluorescence (TILL Photonics). Images were acquired using a TILL Image QE charge-coupled device camera (TILL Photonics) and MetaMorph Software (Molecular Devices). The total internal reflection angle was manually adjusted for every experiment. TIRF time-lapse movies were recorded with 2–5 frames/s. Image analysis was performed in ImageJ.
Chehab T., Santos N.C., Holthenrich A., Koerdt S.N., Disse J., Schuberth C., Nazmi A.R., Neeft M., Koch H., Man K.N., Wojcik S.M., Martin T.F., van der Sluijs P., Brose N, & Gerke V. (2017). A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel–Palade body exocytosis in endothelial cells. Molecular Biology of the Cell, 28(12), 1688-1700.
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3

3D Culture of Breast Cancer Cells

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MCF7 and RD-BCSC cells in 40 μL plug of Matrigel (growth factor reduced and phenol red free, Becton Dickinson, Plymouth, UK) were plated to the well of an 8-well LabTek Chambered coverglass (Nunc, Rochester, USA) at 37°C for 30 min. On ice, cells were prepared at a concentration of 5,000 cells/ml in KSFM supplemented with 5 ng/ml EGF, 2% (v/v) FCS, 4% (v/v) Matrigel, and 0.2 mL of this cell solution was plated onto the Matrigel plug and incubated for 30 min at 37°C, after which 0.2 mL of growth medium was added (KSFM supplemented with 5 ng/mL EGF, 2% (v/v) FCS). Culture medium was changed every 2–3 days. At day 10, morphology was assessed by phase microscopy and cells were fixed and processed for immunofluorescence microscopy analysis.
Han S., Wei R., Zhang X., Jiang N., Fan M., Huang J.H., Xie B., Zhang L., Miao W., Butler A.C., Coleman M.A., Vaughan A.T., Wang Y., Chen H.W., Liu J, & Li J.J. (2019). CPT1A/2-Mediated FAO Enhancement—A Metabolic Target in Radioresistant Breast Cancer. Frontiers in Oncology, 9, 1201.
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4

Tethering Anti-Ig on Planar Lipid Bilayers

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As a surrogate of antigen tethered on APCs, a PLB was used to tether anti-Ig as a model membrane-associated antigen. PLBs were prepared as described previously (51 (link)). Briefly, PLBs were prepared in 8- or 18-well chambered cover glass made by nanostrip-cleaned #1.5 cover glass adhered on it with either 8-well Lab-Tek chambered cover glass (Nunc, 155411) replaced for the bottom coverglass or sticky Slide (ibidi, 81818). A working solution of 100 μM small, unilamellar vesicles was prepared from the stock solution, consisting of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC, 850375) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (DOPE-cap biotin, 870273C) from Avanti Polar Lipids at a ratio of 100:1. To bind anti-Ig to the PLB, the wells containing PLB were incubated at room temperature with streptavidin (2.5 μg/ml) for 10 min, which was followed by incubation with biotinylated goat F(ab′)2 anti-human κ + λ (1 μg/ml) for 20 min with washing between the incubations with PBS.
Horn B.W, & Dorner J.W. (1999). Regional Differences in Production of Aflatoxin B1 and Cyclopiazonic Acid by Soil Isolates of Aspergillus flavus along a Transect within the United States. Applied and Environmental Microbiology, 65(4), 1444-1449.
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5

Western Blot and Immunocytochemistry Analysis

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Proteins (60 µg/well) were separated on 3–8% NuPage Novex Tris/acetate gels (Invitrogen) and electroblotted onto Immobilon PVDF membranes (Millipore). The membranes were blocked and binding of the primary antibodies (ASPM, E2F1 and β‐actin) and HRP‐conjugated or fluorescent secondary antibodies were visualized using the Odyssey Fc imaging system (LI‐COR Biosciences, Lincoln, NE, USA). For immunocytochemistry, cells were grown in eight‐well Lab‐Tek™ Chambered Coverglass (NUNC, Thermo Scientific, Rockford, IL, USA), fixed with paraformaldehyde, permeabilized (Triton‐X) and blocked with bovine serum albumin (BSA). The cells were incubated overnight at 4 °C with the primary antibodies, reblocked and incubated for 1 h with fluorescent secondary antibodies. DNA was stained with DAPI (see supplementary material, Supplementary materials and methods).
Vange P., Bruland T., Beisvag V., Erlandsen S.E., Flatberg A., Doseth B., Sandvik A.K, & Bakke I. (2015). Genome‐wide analysis of the oxyntic proliferative isthmus zone reveals ASPM as a possible gastric stem/progenitor cell marker over‐expressed in cancer. The Journal of Pathology, 237(4), 447-459.
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6

Live-cell Imaging of Cre-mediated Recombination

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SNAP-23fl/+ or SNAP-23fl/- MEFs were plated in a 2-well Nunc Lab-Tek Chambered Coverglass in a volume of 1 ml (50,000 cells/ml). The following day MEFs were infected with MSCV-GFP-Cre virus in presence of Polybrene. The cultures were incubated at 37°C in a 5% CO2 atmosphere and media was changed every other day. The cultures were moved to the microscope 4 days post-infection. Confocal time series images were collected using a Zeiss LSM510 Meta laser scanning confocal microscope equipped with a 20x Plan-apochromat (N.A. 0.8) objective lens and an environmental stage top incubator with temperature, humidity, and CO2 control. The 488 nm laser line from a multi-line Argon laser was used for imaging GFP+ cells with 0.25% of the output power used for excitation intensity. GFP- cells were imaged using differential interference contrast microscopy. Images were acquired every 20 or 30 min over a time period of 30 hr. Each image in the time series was acquired with 0.44 μm X-Y pixel size and a 1.0 μm optical slice thickness, and exported as a video file using Zeiss Zen software and saved as a movie file (.mov) format at 5 frames per second.
Kaul S., Mittal S.K., Feigenbaum L., Kruhlak M.J, & Roche P.A. (2015). Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival. PLoS ONE, 10(2), e0118311.
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7

Cellular Membrane Permeability Assay

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The ability of iGP-1 to cross cellular membranes was evaluated in STHdhQ7 cells cultured at 33°C as described [42] (link). Cells were seeded on collagen-coated, Lab-Tek chambered coverglass (Nunc) 48 h before use. Prior to imaging, cells were cultured for 45 min with 65 nM LysoTracker Red ±250 nM bafilomycin A1. Medium was then replaced with imaging buffer (120 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 20 mM TES, 5 mM NaHCO3, 1.2 mM Na2SO4, 1.5 mM CaCl2, 0.5 mM MgCl2, 2 mM Glucose, and 0.2% BSA, pH 7.4) containing DMSO, 250 nM bafilomycin A1 or 100 µM iGP-1. Cells were imaged on a Nikon Eclipse Ti-PFS inverted microscope equipped with a Cascade 512B camera (Photometrics, Tucson, AZ), a Plan Fluor 100X/1.3 oil objective, an LB-LS17 Xe-arc light source (attenuated), 10–3 excitation and emission filter wheels (Sutter Instruments, Novato, CA), and an MS-2000 linear encoded motorized stage (ASI, Eugene, OR). The filter sets, given as excitation, dichroic mirror, emission in nm/bandwidth, were, for iGP-1 (1 s exposure), 340/26, 409, 460/80 and for LysoTracker Red (100 ms exposure), 543/22, 562, 588/21 (all from Semrock, Rochester, NY). Image acquisition was controlled by NIS Elements 4.0 (Nikon, Melville, NY). Images of cell-free wells containing buffer with DMSO or iGP-1 were used for background subtraction in Image Analyst MKII (Novato, CA).
Orr A.L., Ashok D., Sarantos M.R., Ng R., Shi T., Gerencser A.A., Hughes R.E, & Brand M.D. (2014). Novel Inhibitors of Mitochondrial sn-Glycerol 3-phosphate Dehydrogenase. PLoS ONE, 9(2), e89938.
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8

Live-cell Imaging of Docetaxel Effects

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Cells were plated on Nunc™ Lab-Tek™ chambered coverglass prior to 125 nM docetaxel addition. A layer of mineral oil was layered over the medium to prevent evaporation. Cells were enclosed in a live-cell chamber with regulated temperature, humidity, and pH. Images were taken every 12 minutes up to 72 hours using a Zeiss AxioObserver Z1 Microscope, 20x objective lens.
Mann J., Yang N., Montpetit R., Kirschenman R., Lemieux H, & Goping I.S. (2020). BAD sensitizes breast cancer cells to docetaxel with increased mitotic arrest and necroptosis. Scientific Reports, 10, 355.
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9

TIRF Microscopy Imaging of Transfected HeLa Cells

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HeLa cells were plated on 8-well Lab-Tek chambered coverglass (Nunc) at a density of 1.5 × 104 cells/well the day before transfection. Plasmid DNA were transfected into cells using TransIT-LT1 (Mirus Bio) with the 1:3 DNA-to reagent ratio. The plasmid DNA used in the transfection are mCherry-MAPPER (50 ng/well) and S1R-GFP (50 ng/well). Cells were washed with extracellular buffer (ECB; 125 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 20 mM Hepes, 10 mM glucose, and 1.5 mM CaCl2, pH = 7.4) before imaging and imaged in the ECB. TIRF microscopy imaging experiments were performed at room temperature with a CFI Apo TIRF 100×/1.49 objective on a spinning-disc confocal TIRF microscope custom-built based on a Nikon Eclipse Ti-E inverted microscope (Nikon Instruments) with a HQ2 camera. The microscope was controlled by Micro-Manager software.
Zhemkov V., Ditlev J.A., Lee W.R., Wilson M., Liou J., Rosen M.K, & Bezprozvanny I. (2021). The role of sigma 1 receptor in organization of endoplasmic reticulum signaling microdomains. eLife, 10, e65192.
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10

Real-time Imaging of T Cell Activation

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8 well LabTek chambered coverglass (Nunc) was coated as previously described (12 (link)). For 5C.C7 T cells, surfaces were incubated with photocaged I-Ek-NPE-MCC at 0.5 µg/ml, the nonstimulatory pMHC I-Ek-HB at 3 µg/ml, and anti-MHC class I H-2Kk at 0.5 µg/ml (clone 36-7-5, BD Biosciences). For CD8+ T cells, surfaces were coated with 0.1 µg/ml H-2Kb-NPE-OVA, 1 µg/ml H-2Db-KAVY, and 1 µg/ml ICAM-1. 2×105 T cells were added to each well in imaging medium (RPMI without phenol red medium with 5% FCS and Hepes). In certain experiments, cells were pretreated for 20 min with 0.5 µM wortmannin, 1 µM IC87114, or DMSO (vehicle control) and the drugs were maintained in the medium for the duration of the imaging experiment. 3 s time-lapse series were recorded for 8 min using an inverted fluorescence videomicroscope (Olympus IX-81) equipped with a 150× (NA 1.45) objective lens. Photoactivation was performed using a Mosaic digital diaphragm apparatus (Photonic Instrument) attached to a mercury (HBO) lamp (Olympus). One UV-pulse was performed for 500 ms after 30s of time-lapse recording in order to decage the peptide and deliver the TCR signal. 488 nm or a 564 nm lasers (Melles Griot) were used to image GFP-tagged or RFP-tagged constructs, respectively. MTOC probes (tubulin, centrin) were imaged using epifluorescence illumination, and C1θ using TIRF.
Chauveau A., Le Floc’h A., Bantilan N.S., Koretzky G.A, & Huse M. (2014). Diacylglycerol kinase-α controls T cell polarity by shaping diacylglycerol accumulation at the immune synapse. Science signaling, 7(340), ra82.
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