bead-based flow cytometry mouse inflammation kit  (Becton Dickinson)

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