Journal: Antioxidants

Article Title: Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1

doi: 10.3390/antiox8100496

Figure Lengend Snippet: Effect of whole blood and erythrocyte components on neuronal cell death. ( a , b ) Effect of whole blood incubation on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to blood at indicated amounts (in 5 mL cell culture medium) and durations before analysis (( a ) representative flow cytometry plots; propidium iodide (PI) was detected in the PE channel and annexin V in the FITC channel; ( b ) quantification as % dead cells from total of n = 6 experiments). **** p < 0.0001 control vs. 10 µL blood 1 h, **** p < 0.0001 control vs. 10 µL blood 6 h, **** p < 0.0001 control vs. 1 µL 24 h, **** p < 0.0001 control vs. 10 µL 24 h. ( c , d ) Effect of hemoglobin exposure on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to hemoglobin at indicated concentrations corresponding to the volumes of blood used in ( a , b ) (( c ) representative flow cytometry plots; ( d ) quantification as % dead cells from total of n = 6 experiments). **** p < 0.0001 control vs 4 mM hemoglobin 1 h, *** p = 0.0002 control vs. 0.4 mM hemoglobin 6 h, **** p < 0.0001 control vs 4 mM hemoglobin 6 h, **** p < 0.0001 control vs. 0.4 mM hemoglobin 24 h, *** p = 0.0002 control vs 4 mM hemoglobin 24 h. ( e , f ) Effect of hemin on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to hemin at indicated concentrations corresponding to the volumes of blood used in ( a , b ) (( e ) representative flow cytometry plots; ( f ) quantification as % dead cells from total of n = 6 experiments). p = n.s. for all comparisons.

Article Snippet: Cell supernatant was harvested and analyzed using bead-based flow cytometry as per manufacturer’s instructions (552364, Mouse Inflammation Kit, Becton Dickinson GmbH, Heidelberg, Germany).

Techniques: Incubation, In Vitro, Flow Cytometry, Staining, Cell Culture, Control

Journal: Antioxidants

Article Title: Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1

doi: 10.3390/antiox8100496

Figure Lengend Snippet: Mitochondrial DAMP pathway does not affect neuronal cell death. ( a , b ) Effect of mitochondria isolated from HT22 cells on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to mitochondria isolated from the indicated number of cells (( a ) representative flow cytometry plots; ( b ) quantification as % dead cells from total of n = 6 experiments). p = n.s. for all comparisons. Propidium iodide (PI) was detected in the PE channel and annexin V in the FITC channel. ( c , d ) Effect of mitochondrial DNA isolated from HT22 cells on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to mitochondrial DNA isolated from the indicated number of cells (( c ) representative flow cytometry plots; ( d ) quantification as % dead cells from total of n = 6 experiments). p = n.s. for all comparisons.

Article Snippet: Cell supernatant was harvested and analyzed using bead-based flow cytometry as per manufacturer’s instructions (552364, Mouse Inflammation Kit, Becton Dickinson GmbH, Heidelberg, Germany).

Techniques: Isolation, In Vitro, Flow Cytometry, Staining

Journal: Antioxidants

Article Title: Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1

doi: 10.3390/antiox8100496

Figure Lengend Snippet: Microglial HO-1 expression and viability in response to blood exposure. ( a ) Representative Western blotting shows induction of HO-1 protein expression in BV-2 microglia in response to exposure to 10 µL blood for 24 h. Double bands are due to antibody reactivity against the truncated form of HO-1. Lower panel shows corresponding loading control using total protein staining. ( b ) Representative flow cytometry FSC/SSC dot plots of BV-2 microglia (upper panel) and primary microglia (lower panel) without and with blood exposure (10 µL accordingly). ( c ) Representative light microscopy images of BV-2 microglia and primary microglia before (left panel) and after blood exposure (right panel; 10 µL accordingly; after washing step to remove erythrocytes). Unchanged morphology, size, and granularity of microglia after exposure to blood indicated unchanged viability.

Article Snippet: Cell supernatant was harvested and analyzed using bead-based flow cytometry as per manufacturer’s instructions (552364, Mouse Inflammation Kit, Becton Dickinson GmbH, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Control, Staining, Flow Cytometry, Light Microscopy

Journal: Antioxidants

Article Title: Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1

doi: 10.3390/antiox8100496

Figure Lengend Snippet: Effect of conditioned microglia medium on neuronal cell death and growth. ( a ) Quantification of flow cytometry plots after annexin V/PI staining of HT22 cells incubated with BV-2 microglia medium conditioned with 10 µL blood and then exposed to blood at the indicated amounts. Microglia medium was first conditioned as described in the methods. Conditioned medium was added to HT22 cells and cells were then exposed to blood. The % neuronal dead cells from total of n = 6 experiments. p = n.s. for all comparisons. ( b ) Quantification of flow cytometry plots after annexin V/PI staining of HT22 cells incubated with BV-2 microglia medium incubated with 10 8 sterile latex beads and then exposed to blood at the indicated amounts. Microglia medium was first conditioned with beads as described in the methods. Conditioned medium was added to HT22 cells and cells were then exposed to blood. The % neuronal dead cells from total of n = 3 experiments. * p = 0.0133 control vs. 1 µL blood, ** p = 0.0031 control vs. 10 µL blood. ( c ) Microscopic cell count of neuronal HT22 cells incubated with blood-conditioned microglia medium. Microglia were exposed to the indicated amount of blood for 24 h. Conditioned medium was then added to HT22 cells for 24 h before analysis. * p = 0.0325 untreated medium vs. 10 7 RBC-conditioned medium, *** p = 0.0007 untreated medium vs. 10 8 RBC-conditioned medium, n = 6 experiments. ( d ) Microscopic cell count of neuronal HT22 cells incubated with bead-conditioned microglia medium. Microglia were exposed to the indicated amount of sterile latex beads for 24 h. Conditioned medium was then added to HT22 cells for 24 h before analysis. p = n.s. for all comparisons.

Article Snippet: Cell supernatant was harvested and analyzed using bead-based flow cytometry as per manufacturer’s instructions (552364, Mouse Inflammation Kit, Becton Dickinson GmbH, Heidelberg, Germany).

Techniques: Flow Cytometry, Staining, Incubation, Sterility, Control, Cell Counting

Journal: Antioxidants

Article Title: Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1

doi: 10.3390/antiox8100496

Figure Lengend Snippet: HO-1 dependent shift in microglial cytokine release after blood exposure. ( a ) Representative flow cytometry plots analyzing the cytokine secretion by Hmox1 fl/fl or LyzM-Cre.Hmox1 fl/fl microglia after 30 min of blood and CO exposure, Cytometric Bead Array (CBA) Mouse Inflammation Kit with IL-6, IL-10, MCP-1, IFN-γ, TNF, and IL-12p70 ( b ) Flow cytometric determination of microglial MCP-1 secretion by Hmox1 fl/fl or LyzM-Cre.Hmox1 fl/fl microglia +/− blood and CO 250 ppm, 30 min. ** p = 0.009 for control Hmox1 fl/fl vs. LyzM-Cre.Hmox1 fl/fl , n = 3 experiments. ( c ) Representative flow cytometry plots analyzing the cytokine secretion by Hmox1 fl/fl or LyzM-Cre.Hmox1 fl/fl microglia after 1 h of blood and CO exposure. ( d ) Flow cytometry of microglial MCP-1 secretion in Hmox1 fl/fl or LyzM-Cre.Hmox1 fl/fl microglia +/− blood and CO 250 ppm, 1 h. ** p = 0.001 for control Hmox1 fl/fl vs. LyzM-Cre.Hmox1 fl/fl , * p = 0.05 LyzM-Cre.Hmox1 fl/fl control vs. CO, n = 3 experiments. ( e , f ) Effect of factors released by microglia on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. HT22 cells were exposed to 10 µL blood and cultivated for 24 h in 50% supernatant of cultured microglia isolated from either Hmox fl/fl or LyzM-Cre.Hmox fl/fl mice and conditioned with 10 µL blood exposure for 1 h. (( e ) representative flow cytometry plots (cond = conditioned; SN = supernatant; PI = propidium iodide; propidium iodide (PI) was detected in the PE channel and annexin V in the FITC channel); ( f ) quantification as fold change vs. Hmox fl/fl from total of n = 6 experiments; dead neuronal cells = annexin V and PI-positive cells; ** p = 0.0026).

Article Snippet: Cell supernatant was harvested and analyzed using bead-based flow cytometry as per manufacturer’s instructions (552364, Mouse Inflammation Kit, Becton Dickinson GmbH, Heidelberg, Germany).

Techniques: Flow Cytometry, Control, In Vitro, Staining, Cell Culture, Isolation

Journal: Nature

Article Title: Direct cell reprogramming is a stochastic process amenable to acceleration

doi: 10.1038/nature08592

Figure Lengend Snippet: a, Schematic of experiments. b, Reprogramming of Pre-B cell monoclonal populations measured as the cumulative number of wells that became Nanog-GFP+. n indicates number of populations monitored. Asterisk indicates flow cytometry for GFP detection was performed every 4 weeks. c, Chimeric mice with agouti coat color from iPSCs derived after 12–13 weeks of DOX. d , Heavy chain rearrangements in iPSCs. e , Relative transgene induction levels of monoclonal populations on DOX. Error bars indicate standard deviation (n=3). f , The population averaged doubling time, t d , for each clonal population. Boxes delineate cases where the same clonal population was measured at different times during DOX induction. Lower two lines (green) represent subcloned iPSC lines. Clone labeling: clone #, weeks on DOX (w#), Nanog-GFP >0.5% status (+/−).

Article Snippet: In addition, samples were subjected to the Tdt-mediated dUTP-biotin nick end labeling (TUNEL) test for apoptotic cells by using flow cytometry based MEBSTAIN apoptosis kit (MBL).

Techniques: Flow Cytometry, Derivative Assay, Standard Deviation, Labeling

Journal: Blood Cancer Journal

Article Title: Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma

doi: 10.1038/s41408-021-00554-9

Figure Lengend Snippet: A Control (sh-C) and APOBEC3G knockdown (sh-A3G) MM.1S and H929 cells were evaluated for the presence of micronuclei, using In Vitro MicroFlow Kit (Litron Labs). Images of micronuclei (I) and bar graphs (II) showing percentage of micronuclei are presented. B MM.1S cells were transduced with control shRNA (sh-C) or that targeting A3G (sh-A3G) and following puromycin selection, cultured for 3 weeks. DNA from these and day 0 (baseline control) cells was extracted and the acquisition of new genomic changes in cultured relative to day 0 cells monitored, using whole genome sequencing (30x). Circos plots showing translocations and other genomic changes (I) and bar graphs showing total number of SNVs and indels (II) are presented. C Control and A3G knockdown MM cells were transfected with a plasmid expressing EGFP. EGFP-positive cells were purified, cultured for additional 72 h, genomic DNA isolated, and EGFP sequences amplified using specific primers. The PCR products were cloned into a plasmid vector to transfect into competent E. coli cells and plated on LB agar. Plasmid DNA from 10 colonies/sample were isolated and sequenced to analyze for mutations. Bar graphs show APOBEC-like mutations.

Article Snippet: To evaluate the impact on genomic instability, control and A3G knockdown cells were analyzed for micronuclei, using a flow cytometry-based Micronucleus Assay (MicroFlow kit, Litron Laboratories, New York, USA) as described by us previously [ ].

Techniques: Control, Knockdown, In Vitro, Transduction, shRNA, Selection, Cell Culture, Sequencing, Transfection, Plasmid Preparation, Expressing, Purification, Isolation, Amplification, Clone Assay

Journal: Blood Cancer Journal

Article Title: Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma

doi: 10.1038/s41408-021-00554-9

Figure Lengend Snippet: Control and A3G-overexpressing U266 cells (described in Fig. ) were evaluated for genomic instability. A Following selection, the cells were evaluated for the presence of micronuclei, using In Vitro MicroFlow Kit (Litron Labs). Images of micronuclei (I) and bar graphs showing percentage of micronuclei (II) are presented. B Control and A3G-overexpressing cells described above were transfected with a plasmid expressing EGFP. EGFP-positive cells were purified, cultured for additional 72 h, genomic DNA isolated, and EGFP sequences amplified using specific primers. The PCR products were cloned into a plasmid vector to transfect into competent E. coli cells and plated on LB agar. Plasmid DNA from 10 colonies/sample were isolated and sequenced to analyze for mutations. I. Bar graphs show APOBEC-like mutations as well as total (or overall) mutations; II. Percent GFP + cells were analyzed by flow cytometry at different time points and plotted in the graph.

Article Snippet: To evaluate the impact on genomic instability, control and A3G knockdown cells were analyzed for micronuclei, using a flow cytometry-based Micronucleus Assay (MicroFlow kit, Litron Laboratories, New York, USA) as described by us previously [ ].

Techniques: Control, Selection, In Vitro, Transfection, Plasmid Preparation, Expressing, Purification, Cell Culture, Isolation, Amplification, Clone Assay, Flow Cytometry