Transduction:Article Title: A human iPS cell line for ready-to-use human iAstrocytes that support human neurons
Article Snippet: Quantitation of synapses per length dendrite along MAP-2 positive processes was performed by counting Synaptophysin-positive puncta overlapping MAP2 signal in 20 μm lengths of dendrites (three random regions per image field) in a blinded fashion using FIJI/ImageJ Version 2.1.0/1.53c. .. iAstrocytes were transduced with AAV5 gfaABC1D-lck-GCaMP6f (GFAP promoter-driven membrane targeted GCaMP6f; Addgene 52924 from Baljit Khakh) with 0.4 μl of 7×1012 vg/ml virus per well of a 24-well two weeks prior to imaging (subsequently diluted by half-medium changes every other day), or incubated with 2-5 μM Cal-520 AM (ab171868 Abcam) in 24-well or 96-well plates for 30-60 min. .. Cells in Gibco FluoroBrite DMEM (A1896701 Thermo Fisher) were imaged for 3 min baseline and 3 min more after addition of 50-100 μM ATP, at a 300 ms frame rate and 100 ms exposure time, with 2x2 pixel live binning to reduce file size and improve signal-to-noise, using an APO LWD 40x WI Delts objective and with 470 nm excitation and a GFP 579/26 nm emission filter on a Nikon Widefield Ti2 epifluorescence microscope with a sCMOS, PCO.edge camera and LED Illumination (Lumencore, SpectraX) and live-cell incubator at 37°C and 5% CO 2 .
Article Title: A calcium imaging pipeline to detect and quantify compound-specific effects in human and mouse astrocytes and astrocyte-neuron cocultures
Article Snippet: .. Mouse astrocytes in mono- or cocultures were transduced with AAV2/5-GFAP:GCaMP6f-LCK (Addgene 52924, from Baljit Khakh; 2.5x10 13 vg/mL, used at 0.3 μL (24-well plate), 0.07-0.1 μL (96-well plate), or 0.03 μL (384-well plate) per well) or AAV2/5-GFAP:jRGECO-Lck (plasmid: Addgene 100854, from Douglas Kim & GENIE Project; the synapsin promoter was replaced with a GFAP promoter and virus prepared by the Charité Viral Core Facility; 8.8x10 12 vg/mL, used at 0.1 μL per well in a 96-well plate). .. Neurons in mouse cultures were transduced with AAV2/1-hSyn:jRCaMP1a (Addgene 100848, from Douglas Kim & GENIE Project; 1.9x10 13 , used at 0.005-0.008 μL per well in a 96-well plate) or AAV2/1-hSyn:GCaMP6f (Addgene 100837, from Douglas Kim & GENIE Project; 2.0x10 13 , used at 0.002-0.005 μL per well in a 96-well plate).
Membrane:Article Title: A human iPS cell line for ready-to-use human iAstrocytes that support human neurons
Article Snippet: Quantitation of synapses per length dendrite along MAP-2 positive processes was performed by counting Synaptophysin-positive puncta overlapping MAP2 signal in 20 μm lengths of dendrites (three random regions per image field) in a blinded fashion using FIJI/ImageJ Version 2.1.0/1.53c. .. iAstrocytes were transduced with AAV5 gfaABC1D-lck-GCaMP6f (GFAP promoter-driven membrane targeted GCaMP6f; Addgene 52924 from Baljit Khakh) with 0.4 μl of 7×1012 vg/ml virus per well of a 24-well two weeks prior to imaging (subsequently diluted by half-medium changes every other day), or incubated with 2-5 μM Cal-520 AM (ab171868 Abcam) in 24-well or 96-well plates for 30-60 min. .. Cells in Gibco FluoroBrite DMEM (A1896701 Thermo Fisher) were imaged for 3 min baseline and 3 min more after addition of 50-100 μM ATP, at a 300 ms frame rate and 100 ms exposure time, with 2x2 pixel live binning to reduce file size and improve signal-to-noise, using an APO LWD 40x WI Delts objective and with 470 nm excitation and a GFP 579/26 nm emission filter on a Nikon Widefield Ti2 epifluorescence microscope with a sCMOS, PCO.edge camera and LED Illumination (Lumencore, SpectraX) and live-cell incubator at 37°C and 5% CO 2 .
Virus:Article Title: A human iPS cell line for ready-to-use human iAstrocytes that support human neurons
Article Snippet: Quantitation of synapses per length dendrite along MAP-2 positive processes was performed by counting Synaptophysin-positive puncta overlapping MAP2 signal in 20 μm lengths of dendrites (three random regions per image field) in a blinded fashion using FIJI/ImageJ Version 2.1.0/1.53c. .. iAstrocytes were transduced with AAV5 gfaABC1D-lck-GCaMP6f (GFAP promoter-driven membrane targeted GCaMP6f; Addgene 52924 from Baljit Khakh) with 0.4 μl of 7×1012 vg/ml virus per well of a 24-well two weeks prior to imaging (subsequently diluted by half-medium changes every other day), or incubated with 2-5 μM Cal-520 AM (ab171868 Abcam) in 24-well or 96-well plates for 30-60 min. .. Cells in Gibco FluoroBrite DMEM (A1896701 Thermo Fisher) were imaged for 3 min baseline and 3 min more after addition of 50-100 μM ATP, at a 300 ms frame rate and 100 ms exposure time, with 2x2 pixel live binning to reduce file size and improve signal-to-noise, using an APO LWD 40x WI Delts objective and with 470 nm excitation and a GFP 579/26 nm emission filter on a Nikon Widefield Ti2 epifluorescence microscope with a sCMOS, PCO.edge camera and LED Illumination (Lumencore, SpectraX) and live-cell incubator at 37°C and 5% CO 2 .
Article Title: A calcium imaging pipeline to detect and quantify compound-specific effects in human and mouse astrocytes and astrocyte-neuron cocultures
Article Snippet: .. Mouse astrocytes in mono- or cocultures were transduced with AAV2/5-GFAP:GCaMP6f-LCK (Addgene 52924, from Baljit Khakh; 2.5x10 13 vg/mL, used at 0.3 μL (24-well plate), 0.07-0.1 μL (96-well plate), or 0.03 μL (384-well plate) per well) or AAV2/5-GFAP:jRGECO-Lck (plasmid: Addgene 100854, from Douglas Kim & GENIE Project; the synapsin promoter was replaced with a GFAP promoter and virus prepared by the Charité Viral Core Facility; 8.8x10 12 vg/mL, used at 0.1 μL per well in a 96-well plate). .. Neurons in mouse cultures were transduced with AAV2/1-hSyn:jRCaMP1a (Addgene 100848, from Douglas Kim & GENIE Project; 1.9x10 13 , used at 0.005-0.008 μL per well in a 96-well plate) or AAV2/1-hSyn:GCaMP6f (Addgene 100837, from Douglas Kim & GENIE Project; 2.0x10 13 , used at 0.002-0.005 μL per well in a 96-well plate).
Imaging:Article Title: A human iPS cell line for ready-to-use human iAstrocytes that support human neurons
Article Snippet: Quantitation of synapses per length dendrite along MAP-2 positive processes was performed by counting Synaptophysin-positive puncta overlapping MAP2 signal in 20 μm lengths of dendrites (three random regions per image field) in a blinded fashion using FIJI/ImageJ Version 2.1.0/1.53c. .. iAstrocytes were transduced with AAV5 gfaABC1D-lck-GCaMP6f (GFAP promoter-driven membrane targeted GCaMP6f; Addgene 52924 from Baljit Khakh) with 0.4 μl of 7×1012 vg/ml virus per well of a 24-well two weeks prior to imaging (subsequently diluted by half-medium changes every other day), or incubated with 2-5 μM Cal-520 AM (ab171868 Abcam) in 24-well or 96-well plates for 30-60 min. .. Cells in Gibco FluoroBrite DMEM (A1896701 Thermo Fisher) were imaged for 3 min baseline and 3 min more after addition of 50-100 μM ATP, at a 300 ms frame rate and 100 ms exposure time, with 2x2 pixel live binning to reduce file size and improve signal-to-noise, using an APO LWD 40x WI Delts objective and with 470 nm excitation and a GFP 579/26 nm emission filter on a Nikon Widefield Ti2 epifluorescence microscope with a sCMOS, PCO.edge camera and LED Illumination (Lumencore, SpectraX) and live-cell incubator at 37°C and 5% CO 2 .
Incubation:Article Title: A human iPS cell line for ready-to-use human iAstrocytes that support human neurons
Article Snippet: Quantitation of synapses per length dendrite along MAP-2 positive processes was performed by counting Synaptophysin-positive puncta overlapping MAP2 signal in 20 μm lengths of dendrites (three random regions per image field) in a blinded fashion using FIJI/ImageJ Version 2.1.0/1.53c. .. iAstrocytes were transduced with AAV5 gfaABC1D-lck-GCaMP6f (GFAP promoter-driven membrane targeted GCaMP6f; Addgene 52924 from Baljit Khakh) with 0.4 μl of 7×1012 vg/ml virus per well of a 24-well two weeks prior to imaging (subsequently diluted by half-medium changes every other day), or incubated with 2-5 μM Cal-520 AM (ab171868 Abcam) in 24-well or 96-well plates for 30-60 min. .. Cells in Gibco FluoroBrite DMEM (A1896701 Thermo Fisher) were imaged for 3 min baseline and 3 min more after addition of 50-100 μM ATP, at a 300 ms frame rate and 100 ms exposure time, with 2x2 pixel live binning to reduce file size and improve signal-to-noise, using an APO LWD 40x WI Delts objective and with 470 nm excitation and a GFP 579/26 nm emission filter on a Nikon Widefield Ti2 epifluorescence microscope with a sCMOS, PCO.edge camera and LED Illumination (Lumencore, SpectraX) and live-cell incubator at 37°C and 5% CO 2 .
Plasmid Preparation:Article Title: Midbrain PAG Astrocytes Modulate Mouse Defensive and Panic‐Like Behaviors
Article Snippet: .. Using a syringe pump (Stoelting Co.), a 10‐mL Hamilton syringe (Hamilton Co., Reno, NV, USA) delivered 0.8 mL of pZac2.1‐GfaABC1D‐lck‐GCaMP6f (#52924 plasmid, 52924‐AAV5, Addgene, Watertown, MA, USA) to the dPAG (anteroposterior (AP): 0.00 mm, mediolateral (ML): ±0.40 mm, dorsoventral (DV) ‐2.50 mm from the lambda). ..
Article Title: Astrocytes enable amygdala neural representations supporting memory.
Article Snippet: .. A viral construct containing the genetically engineered Ca2+ indicators AAV5-pZac2.1 gfaABC1D-cyto-GCaMP6f (targeting the astrocyte cytosol75) (titre, 1.8 × 1013 vg per ml, Addgene plasmid 52925; provided by B. Khakh), AAV5-pZac2.1 gfaABC1D-lck-GCaMP6f (targeting astrocyte processes76) (titre, 1.8 × 1013 vg per ml, Addgene plasmid 52924, provided by B. Khakh) or AAV9-hSyn-jGCaMP7f-WPRE (targeting neurons77) (titre 2.5 × 1013 vg per ml, Addgene plasmid 104488, provided by D. Kim) was unilaterally injected (0.45 μl) into the BLA, and BLA-targeting optic fibres were unilaterally implanted, as described above in the ‘General surgical and histological procedures’ section. .. The same procedure was used for simultaneous photometry of astrocytes and neurons, but with AAV5-pZac2.1 gfaABC1D-cyto-GCaMP6f injected into the BLA of one hemisphere and pAAV.Syn.GCaMP6f.WPRE.SV40 (titre, 2.1 × 1013 vg per ml, Addgene plasmid 100837; provided by D. Kim) injected into the BLA of the other hemisphere (counterbalancing which hemisphere received each virus).
Article Title: Midbrain PAG Astrocytes Modulate Mouse Defensive and Panic-Like Behaviors.
Article Snippet: .. Using a syringe pump (Stoelting Co.), a 10-mL Hamilton syringe (Hamilton Co., Reno, NV, USA) delivered 0.8 mL of pZac2.1-GfaABC1D-lckGCaMP6f (#52924 plasmid, 52924-AAV5, Addgene, Watertown, MA, USA) to the dPAG (anteroposterior (AP): 0.00 mm, mediolateral (ML): ± 0.40 mm, dorsoventral (DV) -2.50 mm from the lambda). ..
Article Title: A calcium imaging pipeline to detect and quantify compound-specific effects in human and mouse astrocytes and astrocyte-neuron cocultures
Article Snippet: .. Mouse astrocytes in mono- or cocultures were transduced with AAV2/5-GFAP:GCaMP6f-LCK (Addgene 52924, from Baljit Khakh; 2.5x10 13 vg/mL, used at 0.3 μL (24-well plate), 0.07-0.1 μL (96-well plate), or 0.03 μL (384-well plate) per well) or AAV2/5-GFAP:jRGECO-Lck (plasmid: Addgene 100854, from Douglas Kim & GENIE Project; the synapsin promoter was replaced with a GFAP promoter and virus prepared by the Charité Viral Core Facility; 8.8x10 12 vg/mL, used at 0.1 μL per well in a 96-well plate). .. Neurons in mouse cultures were transduced with AAV2/1-hSyn:jRCaMP1a (Addgene 100848, from Douglas Kim & GENIE Project; 1.9x10 13 , used at 0.005-0.008 μL per well in a 96-well plate) or AAV2/1-hSyn:GCaMP6f (Addgene 100837, from Douglas Kim & GENIE Project; 2.0x10 13 , used at 0.002-0.005 μL per well in a 96-well plate).
Control:
Construct:Article Title: Astrocytes enable amygdala neural representations supporting memory.
Article Snippet: .. A viral construct containing the genetically engineered Ca2+ indicators AAV5-pZac2.1 gfaABC1D-cyto-GCaMP6f (targeting the astrocyte cytosol75) (titre, 1.8 × 1013 vg per ml, Addgene plasmid 52925; provided by B. Khakh), AAV5-pZac2.1 gfaABC1D-lck-GCaMP6f (targeting astrocyte processes76) (titre, 1.8 × 1013 vg per ml, Addgene plasmid 52924, provided by B. Khakh) or AAV9-hSyn-jGCaMP7f-WPRE (targeting neurons77) (titre 2.5 × 1013 vg per ml, Addgene plasmid 104488, provided by D. Kim) was unilaterally injected (0.45 μl) into the BLA, and BLA-targeting optic fibres were unilaterally implanted, as described above in the ‘General surgical and histological procedures’ section. .. The same procedure was used for simultaneous photometry of astrocytes and neurons, but with AAV5-pZac2.1 gfaABC1D-cyto-GCaMP6f injected into the BLA of one hemisphere and pAAV.Syn.GCaMP6f.WPRE.SV40 (titre, 2.1 × 1013 vg per ml, Addgene plasmid 100837; provided by D. Kim) injected into the BLA of the other hemisphere (counterbalancing which hemisphere received each virus).
Article Title: Astrocytes and neurons exhibit partially shared but distinct composite receptive fields for natural stimuli.
Article Snippet: Astrocytes are increasingly recognized as active participants in sensory processing, but whether they show selective responses to stimulus features, analogous to neuronal receptive fields, is not yet established.. To address this, we used two-photon calcium imaging in the auditory cortex of anesthetized mice during presentation of natural ultrasonic vocalizations.. Our aim was to compare astrocytic responses with those of neighboring neurons and to determine whether astrocytes exhibit feature-selective receptive fields.
Injection:Article Title: Astrocytes enable amygdala neural representations supporting memory.
Article Snippet: .. A viral construct containing the genetically engineered Ca2+ indicators AAV5-pZac2.1 gfaABC1D-cyto-GCaMP6f (targeting the astrocyte cytosol75) (titre, 1.8 × 1013 vg per ml, Addgene plasmid 52925; provided by B. Khakh), AAV5-pZac2.1 gfaABC1D-lck-GCaMP6f (targeting astrocyte processes76) (titre, 1.8 × 1013 vg per ml, Addgene plasmid 52924, provided by B. Khakh) or AAV9-hSyn-jGCaMP7f-WPRE (targeting neurons77) (titre 2.5 × 1013 vg per ml, Addgene plasmid 104488, provided by D. Kim) was unilaterally injected (0.45 μl) into the BLA, and BLA-targeting optic fibres were unilaterally implanted, as described above in the ‘General surgical and histological procedures’ section. .. The same procedure was used for simultaneous photometry of astrocytes and neurons, but with AAV5-pZac2.1 gfaABC1D-cyto-GCaMP6f injected into the BLA of one hemisphere and pAAV.Syn.GCaMP6f.WPRE.SV40 (titre, 2.1 × 1013 vg per ml, Addgene plasmid 100837; provided by D. Kim) injected into the BLA of the other hemisphere (counterbalancing which hemisphere received each virus).
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![( A ) HEK293 cells were transfected to express the Ca 2+ sensor ER-GCaMP6-210. Scale bar = 10 μm. ( B ) Resting [Ca 2+ ] ER values were defined as the steady-state fluorescence value after the medium conditioning step and before stimulation with 100 μM ATP + 100 μM CCh. Statistical analysis with unpaired t-test, p < 0.0001. ( C ) Values of [Ca 2+ ] ER after Ca 2+ release stimulated by ATP+CCh. Statistical analysis with unpaired t-test, p < 0.0001. ( D ) Decrease in [Ca 2+ ] ER after ATP+CCh stimulus. This decrease was calculated by subtracting the [Ca 2+ ] ER after stimulation with ATP+CCh from the resting [Ca 2+ ] ER . Statistical analysis with unpaired t-test and p = 0.0471. In ( B – D ), the [Ca 2+ ] ER values were monitored in different ROIs from 4 independent experiments. Total number of ROIs analyzed: n = 62 WT and n = 50 KO ROIs. The black line represents the mean of the data. ( E ) Measurement of [Ca 2+ ] i after Ca 2+ release from the ER in WT and STIM1-KO cells. Representative fluorescence profile of fura-2-loaded cells after stimulation with 100 μM ATP + 100 μM CCh in Ca 2+ -free Hank’s balanced salt solution (min = 1). The graph shows the mean F340/F380 ratio ± S.D. over time from 3 independent experiments for WT HEK293 (black line) and 4 experiments for STIM1-KO cells (red line). Total number of cells analyzed: n = 65 WT and n = 89 KO cells. ( F ) Analysis of the maximum increase in the F340/F380 ratio after ATP+CCh addition in Ca 2+ -free assay medium. The maximum values of the F340/F380 ratio of each cell were normalized relative to the resting ratio value to calculate the magnitude of the increase in [Ca 2+ ] i after ER Ca 2+ release. The mean of the data is represented by the black line. Statistical analysis with unpaired t-test. ( G ) Representative fluorescence image and ( H ) fluorescence profile of mito 4× -GCaMP6f after ATP+CCh stimulus. Cells stably transfected for the expression of the mitochondrial Ca 2+ sensor mito 4 × -GCaMP6f were cultured on collagen-coated coverslips and treated with 1 μg/ml doxycycline to induce expression of the Ca 2+ sensor. ER Ca 2+ release was triggered with ATP+CCh in Ca 2+ -free medium, as in ( E ) and ( F ). The ratios of fluorescence at each time point relative to resting fluorescence (F/F r ) were calculated in different ROIs. The graph shows the mean F/F r ± S.E.M. from 4 independent experiments for WT HEK293 ( n = 39 WT ROIs, black line) and STIM1-KO cells ( n = 40 KO ROIs, red line). ( I ) Analysis of the maximum fluorescence increase (F max /F r ) after ATP+CCh. The F max /F r of all ROIs was analyzed from 15 independent experiments for WT cells and 17 experiments for KO cells ( n = 160 WT and n = 169 KO ROIs). The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001. ( J ) Cells were transiently transfected to express the 4mtD3cpv sensor to measure basal [Ca 2+ ] mit . Data from n = 19 WT cells and n = 16 STIM1-KO cells from 4 independent experiments. The black line represents the mean of data. Statistical analysis with unpaired t-test, p < 0.0001. .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3705/pmc12953705/pmc12953705__44318_2026_700_Fig3_HTML.jpg)
