Retrovirus-mediated and lentivirus-mediated gene transductions were performed as described previously40 (link). To generate HT-LC3 U-2 OS and HeLa cells, cells were transduced with lentiviruses encoding HT-LC3 and selected with 1 µg/ml puromycin for 5 days. ATG2A/B double-knockout and ATG7 knockout U-2 OS cells were generated as previously described39 (link). To generate ATG13 knockout U-2 OS cells, cells were transfected with an equal amount (6 µg each per a 10-cm dish) of three human ATG13 gRNA for 48 h and sorted for GFP-positive transfected cells. Fourteen days after transfection, the cells were re-sorted for GFP-negative population to eliminate Cas9 stable transfectants and used for experiments. For siRNA screening, cells were grown overnight on the Lab-TekII 8-well Chambered Coverglass (Nunc, 155409) and incubated in Accell siRNA Delivery Medium (Dharmacon, B-005000-100) containing 1 µM Accell siRNA for 72 h. siRNA-mediated gene silencing was performed by nucleofection according to the manufacture’s protocol.
Lab tekii 8 well chambered coverglass
Manufactured by Thermo Fisher Scientific
The Lab-TekII 8-well Chambered Coverglass is a laboratory equipment used for cell culture and microscopy applications. It provides an optically clear, sterile, and pre-treated surface for cell attachment and growth. The product features eight individual chambers, allowing for the simultaneous observation and analysis of multiple cell samples.
Automatically generated - may contain errors
3 protocols using lab tekii 8 well chambered coverglass
1
Genetic Manipulations in Cell Lines
2
Metabolic Stress Imaging of Myoblasts
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C2C12 cells (mouse myoblasts, ATCC CRL-1772) were grown in Gibco DMEM/F-12 (1:1) + GlutaMAX medium (ThermoFisher, 31331–028) supplemented by 10% FBS (Biowest, VWR #S1810–500). 24 hours before measurements, cells were seeded in #1.5 glass bottom microscopy wells (Nunc Lab-Tek II 8-well Chambered Coverglass), using the same medium. 30 minutes before measurements, the medium was changed to clear Gibco FluoroBrite DMEM imaging medium (ThermoFisher, A18967–01). The imaging medium contained either no additions (“control cells”), 200 µM dinitrophenol (Sigma D198501) (“DNP-exposed cells”) or 1 mM potassium cyanide (Sigma 60178) (“cyanide-exposed cells”).
3
Aggregation of Citrate-Coated Gold Nanoparticles
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Citrate-coated 5 nm AuNPs were aggregated using benzylmercaptan.77 (link)Note that benzylmercaptan requires handling in an air-circulating fume hood. AuNPs at OD =
1 were mixed with either 12.5 or 25 μM benzylmercaptan at a 1:1 v/v ratio and were
incubated for 30 min at RT on a shaker at 350 rpm. Aggregation of AuNP–antibody
conjugates was triggered by the addition of a ∼1000-fold molar excess of 1 kDa
NHS-PEG-NHS (NanoCS) to the conjugates for 30 min at RT and 24 h at 4 °C. The
absorbance spectra of the aggregated samples were measured using UV–vis
spectrophotometry (Synergy HT, BioTek Instruments). Then, 79.5 μL aliquots of the
samples were mixed with 69 μL of 10.76 mg/mL collagen and 1.5 μL of 1 M NaOH.
The mixtures were loaded into Lab-Tek II 8-well chambered coverglass (Nalge Nunc
International), and the gels were allowed to solidify at 37 °C for 20 min before TP
imaging. Excitation spectra of the aggregated nanoparticles were measured from 740 to 990
nm in 10 nm increments stepped manually using the Mai Tai control software (Spectra
Physics, Mountain View, CA, USA) with the emission detected using a Leica nondescanned
detector with a 560–680 nm bandpass emission filter (see the full system
description below). Emission spectra were measured from 400 to 730 nm with 830 and 880 nm
excitation wavelengths, using the descanned optical path of the Leica SP5MP confocal
spectral scanner.
1 were mixed with either 12.5 or 25 μM benzylmercaptan at a 1:1 v/v ratio and were
incubated for 30 min at RT on a shaker at 350 rpm. Aggregation of AuNP–antibody
conjugates was triggered by the addition of a ∼1000-fold molar excess of 1 kDa
NHS-PEG-NHS (NanoCS) to the conjugates for 30 min at RT and 24 h at 4 °C. The
absorbance spectra of the aggregated samples were measured using UV–vis
spectrophotometry (Synergy HT, BioTek Instruments). Then, 79.5 μL aliquots of the
samples were mixed with 69 μL of 10.76 mg/mL collagen and 1.5 μL of 1 M NaOH.
The mixtures were loaded into Lab-Tek II 8-well chambered coverglass (Nalge Nunc
International), and the gels were allowed to solidify at 37 °C for 20 min before TP
imaging. Excitation spectra of the aggregated nanoparticles were measured from 740 to 990
nm in 10 nm increments stepped manually using the Mai Tai control software (Spectra
Physics, Mountain View, CA, USA) with the emission detected using a Leica nondescanned
detector with a 560–680 nm bandpass emission filter (see the full system
description below). Emission spectra were measured from 400 to 730 nm with 830 and 880 nm
excitation wavelengths, using the descanned optical path of the Leica SP5MP confocal
spectral scanner.
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