rhnull rhmod rbcs  (Worthington Biochemical)


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    Name:
    Hemoglobin
    Description:
    Suitable protease substrate Prepared from repeatedly washed then lysed and dialyzed bovine red cells A lyophilized powder
    Catalog Number:
    LS002402
    Price:
    28
    Source:
    Bovine Erythrocytes
    Size:
    5 gm
    Cas Number:
    9008.02.0
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    Structured Review

    Worthington Biochemical rhnull rhmod rbcs
    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; <t>Rhnull/Rhmod</t> <t>RBCs).</t> b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Suitable protease substrate Prepared from repeatedly washed then lysed and dialyzed bovine red cells A lyophilized powder
    https://www.bioz.com/result/rhnull rhmod rbcs/product/Worthington Biochemical
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhnull rhmod rbcs - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    2) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    3) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    4) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    5) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    6) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    7) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    8) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    9) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    10) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    11) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    12) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    13) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    14) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    15) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    16) Product Images from "NLRP3 Inflammasome Activation Regulates Aged RBC Clearance"

    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

    Journal: Inflammation

    doi: 10.1007/s10753-018-0784-9

    D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.
    Figure Legend Snippet: D-PCR results. a β-Actin concentrations of the 15 samples. Although the mRNA concentration of each sample was adjusted to the same value before the reverse-transcription PCR, the β-actin concentrations differed. The highest concentration was 8500 copies/μL (THP-1 and nigericin), and the lowest was 5980 copies/μL (THP-1; Rhnull/Rhmod RBCs). b NLRP3 results of the THP-1 cells only. Almost all the liquid drops showed negative results. c The NLRP3 results of the THP-1 cells and 42 °C-treated RBCs. The positive and negative findings were equal. d The NLRP3 results of the THP-1 cells and nigericin. Almost all the liquid drops showed positive results. e – i Relative amounts of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ. As the β-actin concentrations differed, the expression levels of NLRP3, IL-1β, IL-6, IL-12p40, and IFNγ were presented as relative amounts gained by dividing the target factor concentrations by the β-actin concentration. The obtained relative amounts were similar to those achieved using the CBA assay. The NLRP3 can be activated, and the mRNA levels of IL-1β, IL-6, IL-12p40, and IFNγ were upregulated by the untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin. The IgG-opsonized RBCs, hemoglobin, and RBC ghosts strongly induced such reactions. When the NLRP3 inhibitor existed in the culture system, the mRNA levels of NLRP3 and cytokines were downregulated. QuantaSoft only showed one result for each detected factor; the p value could not be calculated.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Expressing, Crocin Bleaching Assay, Incubation

    CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p
    Figure Legend Snippet: CD47 expression detected using flow cytometry and immunoblotting. a CD47 expression on normal RBCs ( n = 7), Rhnull/Rhmod RBCs ( n = 1, test in triplicate), and 42 °C-incubated RBCs ( n = 3) tested using flow cytometry. RBCs without any CD47 expression were not reported in humans. The negative result was obtained by the normal RBCs that were reacted directly with the secondary antibodies. The MFI was used to show the CD47 levels. The MFIs of the negative result, normal RBCs, Rhnull/Rhmod RBCs, and 42 °C-incubated RBCs were 55 ± 7, 5938 ± 368, 912 ± 194, and 2909 ± 252, respectively. b CD47 expression on the RBC ghost and normal RBCs detected using immunoblotting. The relative amount obtained by the CD47 light density/GPA light density ratio was used to display the CD47 expression. The CD47 level on the RBC ghosts was similar to that on the normal RBCs. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p
    Figure Legend Snippet: Phagocytosis rates of the different groups of RBCs. The MMA results showed that the phagocytic rate of IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs were 46.00% ± 3.46%, 35.32 ± 5.71%, 15.05% ± 2.07%, and 13.72% ± 2.29%, respectively. The IgG-opsonized RBCs exhibited the highest tendency for clearance by THP-1 cells, whereas the phagocytosis rate of the Rhnull/Rhmod RBCs was not significant compared with which of untreated RBCs. In the presence of the NLRP3 inhibitor in the culture system, the phagocytic rates of the RBCs were downregulated, and the phagocytic rate of the IgG-opsonized RBCs, 42 °C-incubated RBCs, Rhnull/Rhmod RBCs, and untreated RBCs became 25.94% ± 5.79%, 19.46 ± 3.18%, 3.78% ± 1.07%, and 2.54% ± 0.92%, respectively. ** p

    Techniques Used: Incubation

    Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p
    Figure Legend Snippet: Effect of NLRP3 inhibitor detected using CBA assay. The negative control was the THP-1 cells only. The expression of IL-1β or TNFα showed a similar tendency in the 30-min and 4-h reactions. The NLRP3 inhibitor significantly downregulated the expression of the two proinflammatory cytokines in both the 30-min and 4-h reactions. The NLRP3 inhibitor showed a higher inhibition ratio in most groups at 4 h incubation than at 30 min incubation, except in the RBC ghost group in both IL-1β and TNFα productions and in the untreated RBC and Rhnull/Rhmod groups in the TNFα expression. The inhibition rate of the NLRP3 inhibitor was associated with the ability of the stimulus to induce the proinflammatory cytokine production. Nigericin, the IgG-opsonized RBCs, and hemoglobin were the three strongest stimulants to IL-1β secretion. After the 4-h reaction, the expression levels of IL-1β in the three groups were downregulated to 95.0, 70.0, and 59.1%, respectively. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Expressing, Inhibition, Incubation

    IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p
    Figure Legend Snippet: IL-1β and TNFα production detected using the CBA assay. The negative control was the THP-1 cells only. Without considering the reaction time, nigericin (the NLRP3 activation positive control) triggered the THP-1 to produce the most IL-1β. Hemoglobin was the strongest stimulus of TNFα expression in the 30 min incubation. When the reaction was prolonged to 4 h, the cytokine expression evidently increased, and the strongest irritant became nigericin. The Rhnull/Rhmod RBCs exhibited the poorest ability among the RBCs, even less to that of the untreated RBCs, in inducing THP-1 to produce IL-1β or TNFα. * p

    Techniques Used: Crocin Bleaching Assay, Negative Control, Activation Assay, Positive Control, Expressing, Incubation

    Related Articles

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    Article Title: NLRP3 Inflammasome Activation Regulates Aged RBC Clearance
    Article Snippet: After 5 min, each well was added with the untreated RBCs, 42 °C-treated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, hemoglobin (36A16356, Worthington), RBC ghost, and nigericin (XPO424B1014, Sangon) at 1 × 105 RBCs per well.

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