Hela cells were seeded in 8 well dishes (LabTekII, Nalgene) in the conditions previously described (Cell culture & conditions) and transfected with i) a DNA mix containing equal amounts of sCas3-GP (mCitrine-DEVD-mCitrine), sCas9-BP (tBFP-IETD-Cerulean) and sCas8-RP (mCherry-LEHD-mKate) plasmids or ii) with the mCitrine-DEVD-mCitrine-P2A-tagBFP-IETD-Cerulean-T2A-mCherry-LEHD-mKate triple modality reporter construct. 20-24h after transfection, LabTeks were transferred to a Leica TCS SP8 inverted confocal scanning microscope (Leica Microsystems) equipped with a pulsed 470-670 nm white light laser (WLL2 Kit, NKT Photonics), an environment-controlled chamber (Life Imaging Services) and maintained at 37 • C in DMEM (with 25mM HEPES, without Phenol Red). A HC PL APO 63x/1.4NA CS2 oil objective (Leica Microsystems) was used. Fluorescence intensity fluctuations were collected by point-scanning in 10 repetitions of 10 seconds and corresponding ACF (auto-correlation function) curves were fitted to this data.
Tcs sp8 inverted confocal scanning microscope
Manufactured by Leica
The Leica TCS SP8 is an inverted confocal scanning microscope. It is designed to capture high-resolution images of biological samples by scanning them with a focused laser beam and detecting the resulting fluorescence signals.
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2 protocols using tcs sp8 inverted confocal scanning microscope
1
Single-Cell Multiplexed Apoptosis Monitoring
2
Single-Cell Multiplexed Apoptosis Monitoring
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Hela cells were seeded in 8 well dishes (LabTekII, Nalgene) in the conditions previously described (Cell culture & conditions) and transfected with i) a DNA mix containing equal amounts of sCas3-GP (mCitrine-DEVD-mCitrine), sCas9-BP (tBFP-IETD-Cerulean) and sCas8-RP (mCherry-LEHD-mKate) plasmids or ii) with the mCitrine-DEVD-mCitrine-P2A-tagBFP-IETD-Cerulean-T2A-mCherry-LEHD-mKate triple modality reporter construct. 20-24h after transfection, LabTeks were transferred to a Leica TCS SP8 inverted confocal scanning microscope (Leica Microsystems) equipped with a pulsed 470-670 nm white light laser (WLL2 Kit, NKT Photonics), an environment-controlled chamber (Life Imaging Services) and maintained at 37 • C in DMEM (with 25mM HEPES, without Phenol Red). A HC PL APO 63x/1.4NA CS2 oil objective (Leica Microsystems) was used. Fluorescence intensity fluctuations were collected by point-scanning in 10 repetitions of 10 seconds and corresponding ACF (auto-correlation function) curves were fitted to this data.
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