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  • 99
    ATCC fetal bovine serum
    Fetal Bovine Serum, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3087 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum/product/ATCC
    Average 99 stars, based on 3087 article reviews
    Price from $9.99 to $1999.99
    fetal bovine serum - by Bioz Stars, 2020-10
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    99
    Thermo Fisher fetal bovine serum
    Fetal Bovine Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 226538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum/product/Thermo Fisher
    Average 99 stars, based on 226538 article reviews
    Price from $9.99 to $1999.99
    fetal bovine serum - by Bioz Stars, 2020-10
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    99
    Thermo Fisher fetal bovine serum fbs
    Fetal Bovine Serum Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 91565 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum fbs/product/Thermo Fisher
    Average 99 stars, based on 91565 article reviews
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    94
    GE Healthcare fbs
    Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 28650 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/GE Healthcare
    Average 94 stars, based on 28650 article reviews
    Price from $9.99 to $1999.99
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    99
    Millipore fetal bovine serum
    Fetal Bovine Serum, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 44859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum/product/Millipore
    Average 99 stars, based on 44859 article reviews
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    92
    Atlanta Biologicals fbs
    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% <t>FBS/DMEM</t> medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and
    Fbs, supplied by Atlanta Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 5766 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Atlanta Biologicals
    Average 92 stars, based on 5766 article reviews
    Price from $9.99 to $1999.99
    fbs - by Bioz Stars, 2020-10
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    99
    Millipore fetal bovine serum fbs
    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% <t>FBS/DMEM</t> medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and
    Fetal Bovine Serum Fbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16849 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum fbs/product/Millipore
    Average 99 stars, based on 16849 article reviews
    Price from $9.99 to $1999.99
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    99
    GE Healthcare fetal bovine serum fbs
    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% <t>FBS/DMEM</t> medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and
    Fetal Bovine Serum Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 17568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum fbs/product/GE Healthcare
    Average 99 stars, based on 17568 article reviews
    Price from $9.99 to $1999.99
    fetal bovine serum fbs - by Bioz Stars, 2020-10
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    99
    Thermo Fisher heat inactivated fetal bovine serum
    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% <t>FBS/DMEM</t> medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and
    Heat Inactivated Fetal Bovine Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heat inactivated fetal bovine serum/product/Thermo Fisher
    Average 99 stars, based on 24133 article reviews
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    heat inactivated fetal bovine serum - by Bioz Stars, 2020-10
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    99
    Thermo Fisher heat inactivated fetal bovine serum fbs
    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% <t>FBS/DMEM</t> medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and
    Heat Inactivated Fetal Bovine Serum Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heat inactivated fetal bovine serum fbs/product/Thermo Fisher
    Average 99 stars, based on 7928 article reviews
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    92
    Gemini Bio fbs
    Src-homology two-domain-containing phosphatase-2 (SHP-2) activation by Ang II requires AMPK. PAEC were grown to 80% confluence and placed in 0.1% <t>FBS/RPMI</t> overnight. A and B : PAEC were treated with Ang II (10 μM) for the indicated times. Cells were pretreated with or without the AMPK inhibitor compound C (40 μM) for 20 min. A : equal amounts of protein from cell lysates were immunoprecipitated with anti-SHP-2. Immunoprecipitated SHP-2 protein was then subjected to phosphatase assays. B : equal amounts of protein were used for SHP-2 immunoprecipitation and blotted for tyrosine phosphorylation. Lower panel shows densitometry of phospho-SHP-2 bands. C : PAEC were treated with 10 μM Ang II for 5 min before preparation of cell lysates. Equal amounts of protein were used for SHP-2 immunoprecipation. Immunoprecipitated protein was then Western blotted for phospho-AMPK ( top ) or for SHP-2 ( bottom ). Lower bar graph indicates phospho-AMPK band density normalized to SHP-2 band density. All data show means ± SD. *Indicates statistical significance from control, P
    Fbs, supplied by Gemini Bio, used in various techniques. Bioz Stars score: 92/100, based on 2611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Gemini Bio
    Average 92 stars, based on 2611 article reviews
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    fbs - by Bioz Stars, 2020-10
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    99
    GE Healthcare heat inactivated fetal bovine serum
    Src-homology two-domain-containing phosphatase-2 (SHP-2) activation by Ang II requires AMPK. PAEC were grown to 80% confluence and placed in 0.1% <t>FBS/RPMI</t> overnight. A and B : PAEC were treated with Ang II (10 μM) for the indicated times. Cells were pretreated with or without the AMPK inhibitor compound C (40 μM) for 20 min. A : equal amounts of protein from cell lysates were immunoprecipitated with anti-SHP-2. Immunoprecipitated SHP-2 protein was then subjected to phosphatase assays. B : equal amounts of protein were used for SHP-2 immunoprecipitation and blotted for tyrosine phosphorylation. Lower panel shows densitometry of phospho-SHP-2 bands. C : PAEC were treated with 10 μM Ang II for 5 min before preparation of cell lysates. Equal amounts of protein were used for SHP-2 immunoprecipation. Immunoprecipitated protein was then Western blotted for phospho-AMPK ( top ) or for SHP-2 ( bottom ). Lower bar graph indicates phospho-AMPK band density normalized to SHP-2 band density. All data show means ± SD. *Indicates statistical significance from control, P
    Heat Inactivated Fetal Bovine Serum, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 7507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heat inactivated fetal bovine serum/product/GE Healthcare
    Average 99 stars, based on 7507 article reviews
    Price from $9.99 to $1999.99
    heat inactivated fetal bovine serum - by Bioz Stars, 2020-10
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    93
    Biological Industries Inc fbs
    Effect of trypsin on NLP formation. NLP were prepared by adding 1 mM of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 to <t>DMEM</t> containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by <t>FBS</t> or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).
    Fbs, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 93/100, based on 1153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Biological Industries Inc
    Average 93 stars, based on 1153 article reviews
    Price from $9.99 to $1999.99
    fbs - by Bioz Stars, 2020-10
    93/100 stars
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    92
    Biowest SAS fbs
    Count of Vero cells cultured in 10% <t>FBS</t> and 10% <t>HPL.</t> Data are expressed as means±SD of three independent experiments ( P > 0.05).
    Fbs, supplied by Biowest SAS, used in various techniques. Bioz Stars score: 92/100, based on 1540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Biowest SAS
    Average 92 stars, based on 1540 article reviews
    Price from $9.99 to $1999.99
    fbs - by Bioz Stars, 2020-10
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    92
    Biochrom fbs
    In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in <t>RPMI</t> 1640 medium supplemented with 10% <t>FBS</t> and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p
    Fbs, supplied by Biochrom, used in various techniques. Bioz Stars score: 92/100, based on 1846 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Biochrom
    Average 92 stars, based on 1846 article reviews
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    94
    PAA Laboratories fbs
    Effect of incubates of platelets activated by collagen or TRAP-6 in the presence or absence of aspirin on HMEC-1 tube formation. HMEC-1 cells were resuspended in platelet incubates diluted with RPMI 1640 medium + 2% <t>FBS</t> and (1:1) and seeded (40,000 cells/well) on a 48-well plate coated with growth factor-reduced basement matrix extract. Platelet incubates contained human PRP stimulated with collagen (30 µg/ml) or TRAP-6 (30 µM) in the presence or absence of aspirin (100 µM) which were added to the cell suspensions. A ) Tube formation was visualized using time-lapse microscopy, where images were taken in phase every 30 min for 16 h (Cell M software). Images (original magnification, ×10) were taken after 16 h incubation (37°C; 5% CO 2 ) in phase or after the addition of calcein AM. B ) The number of branching points was quantitated after 16 h incubation using ImageJ software. Collagen-stimulated or TRAP-6-stimulated platelets significantly increased HMEC-1 tube formation. This effect was markedly reduced in response to aspirin-treated platelets. Data are shown as means ± sem (1-way ANOVA; n = 6). * P
    Fbs, supplied by PAA Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 1411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/PAA Laboratories
    Average 94 stars, based on 1411 article reviews
    Price from $9.99 to $1999.99
    fbs - by Bioz Stars, 2020-10
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    94
    Corning Life Sciences fetal bovine serum
    Effect of incubates of platelets activated by collagen or TRAP-6 in the presence or absence of aspirin on HMEC-1 tube formation. HMEC-1 cells were resuspended in platelet incubates diluted with RPMI 1640 medium + 2% <t>FBS</t> and (1:1) and seeded (40,000 cells/well) on a 48-well plate coated with growth factor-reduced basement matrix extract. Platelet incubates contained human PRP stimulated with collagen (30 µg/ml) or TRAP-6 (30 µM) in the presence or absence of aspirin (100 µM) which were added to the cell suspensions. A ) Tube formation was visualized using time-lapse microscopy, where images were taken in phase every 30 min for 16 h (Cell M software). Images (original magnification, ×10) were taken after 16 h incubation (37°C; 5% CO 2 ) in phase or after the addition of calcein AM. B ) The number of branching points was quantitated after 16 h incubation using ImageJ software. Collagen-stimulated or TRAP-6-stimulated platelets significantly increased HMEC-1 tube formation. This effect was markedly reduced in response to aspirin-treated platelets. Data are shown as means ± sem (1-way ANOVA; n = 6). * P
    Fetal Bovine Serum, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 94/100, based on 2514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum/product/Corning Life Sciences
    Average 94 stars, based on 2514 article reviews
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    94
    Mediatech fbs
    Uptake of B. anthracis spores into mammalian cells cultured under germinating or non-germinating conditions . RAW264.7 cells (A, D), MH-S cells (B, E), or JAWSII cells (C, F) were incubated with B. anthracis spores (MOI 10) in <t>DMEM,</t> RPMI, or DMEM, respectively, in the presence (+, black bars) or absence (-, white bars) of <t>FBS</t> (10%), and then evaluated at 5 or 60 min by flow cytometry and in the presence of trypan blue (0.5%) for the percentage of cells with intracellular spores (A-C), and, for total cell associated spore fluorescence (D-F), as described under Materials and Methods. (A-C) The data are rendered as the percentage of infected cells with the entire population that has internalized spores. (D-F) The data are expressed as the change in MFI, normalized to cells at 5 min post infection in FBS-free medium. To generate the bar graphs, data were combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences in percent infected cells (A) or total intracellular spores (B) between cells incubated in the absence or presence of FBS.
    Fbs, supplied by Mediatech, used in various techniques. Bioz Stars score: 94/100, based on 600 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Mediatech
    Average 94 stars, based on 600 article reviews
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    fbs - by Bioz Stars, 2020-10
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    93
    Wisent Corporation fbs
    Effect of insulin-like growth factor (IGF)-1 on alkaline phosphatase activity in osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 nM 1,25(OH) 2 D 3 in Ham <t>F12/DMEM</t> media containing 2% charcoal-treated <t>FBS</t> and in the presence or not of 50 ng/ml IGF-1 with or without 10 μM PD98059 (PD). Results are expressed as the mean ± SEM of control values without IGF-1 for 6 OA osteoblast preparations.
    Fbs, supplied by Wisent Corporation, used in various techniques. Bioz Stars score: 93/100, based on 1180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Wisent Corporation
    Average 93 stars, based on 1180 article reviews
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    fbs - by Bioz Stars, 2020-10
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    94
    EuroClone fbs
    Proliferation kinetics of UCMSCs in <t>CBS</t> and <t>FBS</t> supplemented media. Initial densities at each passage were 10,000/well and results are shown as means±SE. (A) Population doublings time (PDT). (B) Fold increase. *p-value
    Fbs, supplied by EuroClone, used in various techniques. Bioz Stars score: 94/100, based on 1035 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/EuroClone
    Average 94 stars, based on 1035 article reviews
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    94
    Omega Scientific Inc fbs
    Lipid raft and cholesterol content in T cells cultured in normal or <t>delipidated</t> medium. T cells isolated from naïve apoE-/- mice were cultured in culture medium supplemented with 10% delipidated <t>FBS</t> or 10% FBS medium for 24 hours with CD3/CD28 activation beads. More unesterified cholesterol contents (A) or lipid rafts (B) were observed in activated CD4 + and CD8 + T cells cultured in 10% FBS medium when compared to T cells cultured in 10% delipidated FBS medium. Experiments were repeated 4 times using T cells from 3 mice.
    Fbs, supplied by Omega Scientific Inc, used in various techniques. Bioz Stars score: 94/100, based on 888 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Omega Scientific Inc
    Average 94 stars, based on 888 article reviews
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    Image Search Results


    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% FBS/DMEM medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% FBS/DMEM medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: Transfection, Cell Culture, Luciferase

    l -C14 carnitine induced reactive oxygen species (ROS) but not mitochondrial ROS in murine monocyte/macrophages. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with LPS (100 ng/mL), 25 μM l -C14 acylcarnitine ( l -C14 carn),

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: l -C14 carnitine induced reactive oxygen species (ROS) but not mitochondrial ROS in murine monocyte/macrophages. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with LPS (100 ng/mL), 25 μM l -C14 acylcarnitine ( l -C14 carn),

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques:

    Induction of COX-2 and proinflammatory cytokines are reduced in MyD88 knock-down RAW 264.7 cells. Stable MyD88 KD cells were generated by lentiviral mediated shRNA in RAW 264.7 cells. Cells were then serum-starved for 4–6 h (0.25% FBS/DMEM) then

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: Induction of COX-2 and proinflammatory cytokines are reduced in MyD88 knock-down RAW 264.7 cells. Stable MyD88 KD cells were generated by lentiviral mediated shRNA in RAW 264.7 cells. Cells were then serum-starved for 4–6 h (0.25% FBS/DMEM) then

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: Generated, shRNA

    l -C14 carnitine increases phosphorylation of ERK and JNK in a time-dependent manner: MAP kinases and NF-κB contribution to l -C14 carnitine induced cytokine production. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: l -C14 carnitine increases phosphorylation of ERK and JNK in a time-dependent manner: MAP kinases and NF-κB contribution to l -C14 carnitine induced cytokine production. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques:

    Effect of 2Py on survivin, Bcl-2, and Bax in LNCaP, C4-2, PC-3 and DU 145 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and western blotting analysis performed using antibodies to survivin (n=4), Bax (n=3), and Bcl-2 (n=3), with β-actin as control.

    Journal: Anti-cancer drugs

    Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

    doi: 10.1097/CAD.0b013e328336f57b

    Figure Lengend Snippet: Effect of 2Py on survivin, Bcl-2, and Bax in LNCaP, C4-2, PC-3 and DU 145 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and western blotting analysis performed using antibodies to survivin (n=4), Bax (n=3), and Bcl-2 (n=3), with β-actin as control.

    Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

    Techniques: Cell Culture, Isolation, Western Blot

    Cell number as assessed by crystal violet assay in LNCaP, C4-2 and PC-3 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO, 10, or 25 µM DHA, ON-2Py or 2Py for 24, 48, and 72 h. Relative cell number was assessed by crystal violet assay (n = 4). Results are expressed as the mean ± SD. * indicates significant difference from control (p

    Journal: Anti-cancer drugs

    Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

    doi: 10.1097/CAD.0b013e328336f57b

    Figure Lengend Snippet: Cell number as assessed by crystal violet assay in LNCaP, C4-2 and PC-3 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO, 10, or 25 µM DHA, ON-2Py or 2Py for 24, 48, and 72 h. Relative cell number was assessed by crystal violet assay (n = 4). Results are expressed as the mean ± SD. * indicates significant difference from control (p

    Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

    Techniques: Crystal Violet Assay, Cell Culture

    Effect of 2Py on apoptosis and cell cycle in LNCaP, C4-2, PC-3 and DU 145 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h. The cells were trypsinized and stained with propidium iodide and analyzed on a flow cytometer. The percentage of apoptotic events (Panel A), cell viability (Panel B), and the percentage of cells in G0 and G2M and S phase of the cell cycle (Panels C, D and E) were determined (n=3). Histograms of cell cycle distribution in control and 2Py treated cells (Panel F).

    Journal: Anti-cancer drugs

    Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

    doi: 10.1097/CAD.0b013e328336f57b

    Figure Lengend Snippet: Effect of 2Py on apoptosis and cell cycle in LNCaP, C4-2, PC-3 and DU 145 cells Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h. The cells were trypsinized and stained with propidium iodide and analyzed on a flow cytometer. The percentage of apoptotic events (Panel A), cell viability (Panel B), and the percentage of cells in G0 and G2M and S phase of the cell cycle (Panels C, D and E) were determined (n=3). Histograms of cell cycle distribution in control and 2Py treated cells (Panel F).

    Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

    Effect of 2Py on C-Myc, Cyclin D1, AR, PSA, and β-catenin expression in LNCaP and C4-2 cells (A) Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and Western blotting analysis performed using antibodies to Cyclin D1 (n=3), c-Myc (n=3), AR (n=3), PSA (n=3), and β-catenin (n=2) with β-actin as control (n=3). (B) Immunocytochemical analysis of β-catenin in LNCaP and C4-2 cells cultured in 10% FBS medium with DMSO or 15 µM 2Py for 24 h. Arrows indicate cytoplasmic relocalization of β-catenin after 2Py treatment.

    Journal: Anti-cancer drugs

    Article Title: Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

    doi: 10.1097/CAD.0b013e328336f57b

    Figure Lengend Snippet: Effect of 2Py on C-Myc, Cyclin D1, AR, PSA, and β-catenin expression in LNCaP and C4-2 cells (A) Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and Western blotting analysis performed using antibodies to Cyclin D1 (n=3), c-Myc (n=3), AR (n=3), PSA (n=3), and β-catenin (n=2) with β-actin as control (n=3). (B) Immunocytochemical analysis of β-catenin in LNCaP and C4-2 cells cultured in 10% FBS medium with DMSO or 15 µM 2Py for 24 h. Arrows indicate cytoplasmic relocalization of β-catenin after 2Py treatment.

    Article Snippet: After treatment in RPMI 1640 with 10% fetal bovine serum (FBS) (Atlanta Biologicals) with DMSO or 15 µM 2Py for 48 h, cells were fixed and incubated with rabbit polyclonal anti-human β-catenin antibody (5 µg/mL Santa Cruz Biotechnology Inc. Santa Cruz, CA) or control rabbit IgG (5 µg/mL Vector laboratories Inc. Burlingame, CA) and then with goat-anti-rabbit Alexa Fluor 488 at a dilution of 1:400 respectively, and mounted with ProlongGold anti-fade reagent w/DAPI (Invitrogen Corp.).

    Techniques: Expressing, Cell Culture, Isolation, Western Blot

    Src-homology two-domain-containing phosphatase-2 (SHP-2) activation by Ang II requires AMPK. PAEC were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight. A and B : PAEC were treated with Ang II (10 μM) for the indicated times. Cells were pretreated with or without the AMPK inhibitor compound C (40 μM) for 20 min. A : equal amounts of protein from cell lysates were immunoprecipitated with anti-SHP-2. Immunoprecipitated SHP-2 protein was then subjected to phosphatase assays. B : equal amounts of protein were used for SHP-2 immunoprecipitation and blotted for tyrosine phosphorylation. Lower panel shows densitometry of phospho-SHP-2 bands. C : PAEC were treated with 10 μM Ang II for 5 min before preparation of cell lysates. Equal amounts of protein were used for SHP-2 immunoprecipation. Immunoprecipitated protein was then Western blotted for phospho-AMPK ( top ) or for SHP-2 ( bottom ). Lower bar graph indicates phospho-AMPK band density normalized to SHP-2 band density. All data show means ± SD. *Indicates statistical significance from control, P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms

    doi: 10.1152/ajplung.00072.2011

    Figure Lengend Snippet: Src-homology two-domain-containing phosphatase-2 (SHP-2) activation by Ang II requires AMPK. PAEC were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight. A and B : PAEC were treated with Ang II (10 μM) for the indicated times. Cells were pretreated with or without the AMPK inhibitor compound C (40 μM) for 20 min. A : equal amounts of protein from cell lysates were immunoprecipitated with anti-SHP-2. Immunoprecipitated SHP-2 protein was then subjected to phosphatase assays. B : equal amounts of protein were used for SHP-2 immunoprecipitation and blotted for tyrosine phosphorylation. Lower panel shows densitometry of phospho-SHP-2 bands. C : PAEC were treated with 10 μM Ang II for 5 min before preparation of cell lysates. Equal amounts of protein were used for SHP-2 immunoprecipation. Immunoprecipitated protein was then Western blotted for phospho-AMPK ( top ) or for SHP-2 ( bottom ). Lower bar graph indicates phospho-AMPK band density normalized to SHP-2 band density. All data show means ± SD. *Indicates statistical significance from control, P

    Article Snippet: Passage 2–8 cells were used for all experiments and were cultured in RPMI 1640 medium containing 10% FBS (Gemini Bioproducts, Woodland, CA), 1% penicillin/streptomycin, and 0.5% fungizone (Invitrogen, Carlsbad, CA).

    Techniques: Activation Assay, Immunoprecipitation, Western Blot

    Increase of ATP production induced by angiotensin type 2 receptor (AT 2 ) stimulation. Pulmonary artery endothelial cells (PAEC) were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight before treatment with angiotensin II (Ang II) (10 μM). Relative ATP levels were measured. A : Ang II was added for the indicated times. B : Ang II-induced ATP productions were assayed after 2 h in the presence and absence of compound C (40 μM) or oligomycin (50 nM). Compound C or oligomycin was added 20 min before Ang II. C : Ang II AT 2 receptor antagonist PD123319 (50 μM) was added 20 min before the addition of Ang II for 2 h. All data show means ± SD. * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms

    doi: 10.1152/ajplung.00072.2011

    Figure Lengend Snippet: Increase of ATP production induced by angiotensin type 2 receptor (AT 2 ) stimulation. Pulmonary artery endothelial cells (PAEC) were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight before treatment with angiotensin II (Ang II) (10 μM). Relative ATP levels were measured. A : Ang II was added for the indicated times. B : Ang II-induced ATP productions were assayed after 2 h in the presence and absence of compound C (40 μM) or oligomycin (50 nM). Compound C or oligomycin was added 20 min before Ang II. C : Ang II AT 2 receptor antagonist PD123319 (50 μM) was added 20 min before the addition of Ang II for 2 h. All data show means ± SD. * P

    Article Snippet: Passage 2–8 cells were used for all experiments and were cultured in RPMI 1640 medium containing 10% FBS (Gemini Bioproducts, Woodland, CA), 1% penicillin/streptomycin, and 0.5% fungizone (Invitrogen, Carlsbad, CA).

    Techniques:

    ATP generation and AMPK are required for Ang II-induced apoptosis in PAEC. A and B : PAEC were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight. Neutral comet assays were performed to detect apoptotic cells. Percentage of apoptotic cells were then determined. A : cells were treated with oligomycin (50 nM) for 1 h in glucose-free medium (RPMI, 0% FBS). Cells were washed twice in 10% FBS/RPMI before the addition of Ang II (10 μM) for 16 h. B : cells were treated with and without the AMPK inhibitor compound C (40 μM). C : mitochondria-free cell lysates were Western blotted for active cytochrome c and caspase 3. Blots were stripped and probed for β-actin as a loading control. Representative results are shown. Bar graphs represent normalized band densities for both cytochrome c and activated caspase 3. All bar graphs show means ± SD. * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms

    doi: 10.1152/ajplung.00072.2011

    Figure Lengend Snippet: ATP generation and AMPK are required for Ang II-induced apoptosis in PAEC. A and B : PAEC were grown to 80% confluence and placed in 0.1% FBS/RPMI overnight. Neutral comet assays were performed to detect apoptotic cells. Percentage of apoptotic cells were then determined. A : cells were treated with oligomycin (50 nM) for 1 h in glucose-free medium (RPMI, 0% FBS). Cells were washed twice in 10% FBS/RPMI before the addition of Ang II (10 μM) for 16 h. B : cells were treated with and without the AMPK inhibitor compound C (40 μM). C : mitochondria-free cell lysates were Western blotted for active cytochrome c and caspase 3. Blots were stripped and probed for β-actin as a loading control. Representative results are shown. Bar graphs represent normalized band densities for both cytochrome c and activated caspase 3. All bar graphs show means ± SD. * P

    Article Snippet: Passage 2–8 cells were used for all experiments and were cultured in RPMI 1640 medium containing 10% FBS (Gemini Bioproducts, Woodland, CA), 1% penicillin/streptomycin, and 0.5% fungizone (Invitrogen, Carlsbad, CA).

    Techniques: Western Blot

    Effect of trypsin on NLP formation. NLP were prepared by adding 1 mM of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).

    Journal: PLoS ONE

    Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    doi: 10.1371/journal.pone.0004417

    Figure Lengend Snippet: Effect of trypsin on NLP formation. NLP were prepared by adding 1 mM of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).

    Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

    Techniques: Incubation, Inhibition, SDS Page, Centrifugation

    Energy-dispersive X-ray spectroscopy of NLP and NB. EDX results were selected to illustrate the wide spectrum of NLP compositions, with respect to the amounts of calcium and phosphorus incorporated, and their similarities to NB. (A) Elemental composition of NLP prepared by adding CaCl 2 and (NH 4 ) 2 CO 3 at 50 mM into DMEM corresponding to calcium carbonate. (B and C) Amorphous CaCO 3 NLP produced as in (A) except that the precipitating reagents were added to DMEM and incubated at room temperature for 2 days (B) or one month (C), showing increased incorporation of P with prolonged incubation. Ca/P ratios are 5.09 for (B) and 1.60 for (C), with the latter close to the theoretical value of 1.67 associated with stoichiometric HAP. (D–H) Formation of NLP with various inputs of ions, including P, yielding different Ca/P ratios. (D) NLP formed from 0.25 M each of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 , mixed in with DMEM at vol. ratios of 2∶5∶5∶13 that had been incubated in DMEM for 2 days, revealing the presence of magnesium ions and a Ca/P ratio of 0.78. (E–H) NLP were prepared like in (D) at vol. ratios of (E) 1∶1∶1∶22, yielding a Ca/P ratio of 1.29; (F) 3∶2∶2∶43, Ca/P ratio of 1.49; (G) 9∶5∶5∶106, Ca/P ratio of 1.76; (H) 2∶1∶1∶21, Ca/P ratio of 2.03. NLP were submitted to EDX immediately after formation. (I–L) Elemental composition of various NB preparations. (I and J) NB obtained from one-month-old DMEM cultures containing (I) 10% HS or (J) 10% FBS. Ca/P ratios: 1.75 (I) and 1.53 (J). (K) NB strain DSM 5819, after one week in DMEM without serum, giving a Ca/P ratio of 1.45. (L) “Nanons” after one week in DMEM; Ca/P ratio: 1.66.

    Journal: PLoS ONE

    Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    doi: 10.1371/journal.pone.0004417

    Figure Lengend Snippet: Energy-dispersive X-ray spectroscopy of NLP and NB. EDX results were selected to illustrate the wide spectrum of NLP compositions, with respect to the amounts of calcium and phosphorus incorporated, and their similarities to NB. (A) Elemental composition of NLP prepared by adding CaCl 2 and (NH 4 ) 2 CO 3 at 50 mM into DMEM corresponding to calcium carbonate. (B and C) Amorphous CaCO 3 NLP produced as in (A) except that the precipitating reagents were added to DMEM and incubated at room temperature for 2 days (B) or one month (C), showing increased incorporation of P with prolonged incubation. Ca/P ratios are 5.09 for (B) and 1.60 for (C), with the latter close to the theoretical value of 1.67 associated with stoichiometric HAP. (D–H) Formation of NLP with various inputs of ions, including P, yielding different Ca/P ratios. (D) NLP formed from 0.25 M each of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 , mixed in with DMEM at vol. ratios of 2∶5∶5∶13 that had been incubated in DMEM for 2 days, revealing the presence of magnesium ions and a Ca/P ratio of 0.78. (E–H) NLP were prepared like in (D) at vol. ratios of (E) 1∶1∶1∶22, yielding a Ca/P ratio of 1.29; (F) 3∶2∶2∶43, Ca/P ratio of 1.49; (G) 9∶5∶5∶106, Ca/P ratio of 1.76; (H) 2∶1∶1∶21, Ca/P ratio of 2.03. NLP were submitted to EDX immediately after formation. (I–L) Elemental composition of various NB preparations. (I and J) NB obtained from one-month-old DMEM cultures containing (I) 10% HS or (J) 10% FBS. Ca/P ratios: 1.75 (I) and 1.53 (J). (K) NB strain DSM 5819, after one week in DMEM without serum, giving a Ca/P ratio of 1.45. (L) “Nanons” after one week in DMEM; Ca/P ratio: 1.66.

    Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

    Techniques: Spectroscopy, Produced, Incubation

    Effect of serum, temperature, and EDTA on NLP formation. NLP were prepared from 3 mM of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 in DMEM containing different amounts of FBS or HS, as indicated, followed by incubation at 37°C for 1 day (A and B), 1 week (E and F), or 4°C for 2 weeks (C and D). Note that FBS had a stronger, dose-dependent inhibitory effect on NLP formation in all 3 situations when compared with HS. Inhibition could be seen for 2 weeks at 4°C, but at 37°C, it was overcome after 1 week, and this release of inhibition was more evident with HS. (G ) NLP were prepared exactly as in Fig. 1B , by adding solutions of 0.25 M CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 (pH 7.4) to DMEM to the indicated concentrations in the presence of 10 mM EDTA, followed by incubation for 1 day. Addition of EDTA into the precipitation mix decreased the amount of NLP, except for well 6, containing 10 mM of precipitating reagents, indicating that excess EDTA is needed for the inhibition to work.

    Journal: PLoS ONE

    Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    doi: 10.1371/journal.pone.0004417

    Figure Lengend Snippet: Effect of serum, temperature, and EDTA on NLP formation. NLP were prepared from 3 mM of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 in DMEM containing different amounts of FBS or HS, as indicated, followed by incubation at 37°C for 1 day (A and B), 1 week (E and F), or 4°C for 2 weeks (C and D). Note that FBS had a stronger, dose-dependent inhibitory effect on NLP formation in all 3 situations when compared with HS. Inhibition could be seen for 2 weeks at 4°C, but at 37°C, it was overcome after 1 week, and this release of inhibition was more evident with HS. (G ) NLP were prepared exactly as in Fig. 1B , by adding solutions of 0.25 M CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 (pH 7.4) to DMEM to the indicated concentrations in the presence of 10 mM EDTA, followed by incubation for 1 day. Addition of EDTA into the precipitation mix decreased the amount of NLP, except for well 6, containing 10 mM of precipitating reagents, indicating that excess EDTA is needed for the inhibition to work.

    Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

    Techniques: Incubation, Inhibition

    Protein profiles of NLP and NB as determined by their passage through serum-free or serum-containing medium. (A) Protein profile of HS NB maintained in DMEM containing 5% HS (lane 1) followed by transfer to serum-free DMEM and incubation for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), after which proteins were analyzed by SDS-PAGE. A gradual disappearance of proteins bands is seen with increased incubation time in serum-free medium. (B) Protein profile of NB strain DSM 5821 after 1 day in DMEM containing 2% FBS (lane 1), followed by transfer to serum-free DMEM for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), again showing a gradual disappearance of bands. (C) Protein profiles of NB obtained from 10% HS as in Fig. 3 (lane 1) and HS NLP formed as in Fig. 3 except that 5% HS was present in the precipitating mixture (lane 2). HS NLP were then transferred to DMEM containing 5% HS and incubated for the following periods of time: 1 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). A gradual increase of bands of low molecular weight could be seen along with a fading of the high molecular weight bands, especially the 70–72 kDa band. (D) Protein profiles of NB strain DSM 5820 maintained in 10% γ-irradiated FBS (lane 1) and FBS NLP obtained as in Fig. 3 , except that 5% FBS was added to the precipitating mixture (lane 2). FBS NLP were inoculated into DMEM containing 5% FBS and incubated for the following periods of time: 2 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). By day 14, the protein profile of these FBS NLP, with an increase in the number of bands and a loss of the 70–72 kDa band, closely resembled that of DSM 5820 (lane 1).

    Journal: PLoS ONE

    Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    doi: 10.1371/journal.pone.0004417

    Figure Lengend Snippet: Protein profiles of NLP and NB as determined by their passage through serum-free or serum-containing medium. (A) Protein profile of HS NB maintained in DMEM containing 5% HS (lane 1) followed by transfer to serum-free DMEM and incubation for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), after which proteins were analyzed by SDS-PAGE. A gradual disappearance of proteins bands is seen with increased incubation time in serum-free medium. (B) Protein profile of NB strain DSM 5821 after 1 day in DMEM containing 2% FBS (lane 1), followed by transfer to serum-free DMEM for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), again showing a gradual disappearance of bands. (C) Protein profiles of NB obtained from 10% HS as in Fig. 3 (lane 1) and HS NLP formed as in Fig. 3 except that 5% HS was present in the precipitating mixture (lane 2). HS NLP were then transferred to DMEM containing 5% HS and incubated for the following periods of time: 1 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). A gradual increase of bands of low molecular weight could be seen along with a fading of the high molecular weight bands, especially the 70–72 kDa band. (D) Protein profiles of NB strain DSM 5820 maintained in 10% γ-irradiated FBS (lane 1) and FBS NLP obtained as in Fig. 3 , except that 5% FBS was added to the precipitating mixture (lane 2). FBS NLP were inoculated into DMEM containing 5% FBS and incubated for the following periods of time: 2 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). By day 14, the protein profile of these FBS NLP, with an increase in the number of bands and a loss of the 70–72 kDa band, closely resembled that of DSM 5820 (lane 1).

    Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

    Techniques: Incubation, SDS Page, Molecular Weight, Irradiation

    Protein profiles of NLP and NB cultured from serum. (A) Protein profiles of (A) NB cultured from HS and DMEM, as in Fig. 3 ; (B) NB strain DSM 5819 subcultured in serum-free DMEM for 2 days; (C) FBS NLP prepared as in Fig. 3 in the presence of 5% FBS; (D) HS NLP prepared as in Fig. 3 in the presence of 5% HS. (E) NB as in (A) after three 2-day serial passages through serum-free DMEM. (F) Lane 1: “Nanons” after two 2-day passages in serum-free DMEM. Lane 2: “Nanons” after two 2-day passages in DMEM containing 10% FBS. Gels were stained with Coomassie blue. The protein samples loaded onto each lane and their preparation are described in the Materials and Methods .

    Journal: PLoS ONE

    Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    doi: 10.1371/journal.pone.0004417

    Figure Lengend Snippet: Protein profiles of NLP and NB cultured from serum. (A) Protein profiles of (A) NB cultured from HS and DMEM, as in Fig. 3 ; (B) NB strain DSM 5819 subcultured in serum-free DMEM for 2 days; (C) FBS NLP prepared as in Fig. 3 in the presence of 5% FBS; (D) HS NLP prepared as in Fig. 3 in the presence of 5% HS. (E) NB as in (A) after three 2-day serial passages through serum-free DMEM. (F) Lane 1: “Nanons” after two 2-day passages in serum-free DMEM. Lane 2: “Nanons” after two 2-day passages in DMEM containing 10% FBS. Gels were stained with Coomassie blue. The protein samples loaded onto each lane and their preparation are described in the Materials and Methods .

    Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

    Techniques: Cell Culture, Staining

    Culture of NB from serum and formation of NLP. (A) Culture of NB from FBS (top panel) and HS (bottom panel). Serum was inoculated into DMEM to the indicated concentrations. After incubating for 1 month at 37°C, NB were detected by visual inspection and A 650 (insets). For FBS, the amount of NB was maximal at 0.3%, decreasing thereafter with higher FBS concentrations, while for HS, NB were only visually noticeable at HS concentrations exceeding 3%. (B) Formation of NLP in DMEM. NLP were prepared by successively adding solutions of 0.25 M CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 (pH 7.4) into DMEM as indicated and incubated at 37°C overnight, which resulted in dose-dependent formation of NLP (top row). The same experiment was repeated in the presence of 1% FBS, which showed inhibition of NLP formation (bottom row). This serum inhibition was overcome by increased concentrations of precipitating reagents, at 3 mM and above.

    Journal: PLoS ONE

    Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    doi: 10.1371/journal.pone.0004417

    Figure Lengend Snippet: Culture of NB from serum and formation of NLP. (A) Culture of NB from FBS (top panel) and HS (bottom panel). Serum was inoculated into DMEM to the indicated concentrations. After incubating for 1 month at 37°C, NB were detected by visual inspection and A 650 (insets). For FBS, the amount of NB was maximal at 0.3%, decreasing thereafter with higher FBS concentrations, while for HS, NB were only visually noticeable at HS concentrations exceeding 3%. (B) Formation of NLP in DMEM. NLP were prepared by successively adding solutions of 0.25 M CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 (pH 7.4) into DMEM as indicated and incubated at 37°C overnight, which resulted in dose-dependent formation of NLP (top row). The same experiment was repeated in the presence of 1% FBS, which showed inhibition of NLP formation (bottom row). This serum inhibition was overcome by increased concentrations of precipitating reagents, at 3 mM and above.

    Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

    Techniques: Incubation, Inhibition

    Protein identification in NLP prepared from FBS and HS. Protein profiles correspond to Figs. 11C and D , reproduced here to illustrate the protein identification procedure used as well as the results obtained. NLP were prepared from DMEM containing 5% FBS or 5% HS that had been precipitated with 10 mM each of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 . Following SDS-PAGE and Coomassie blue staining, the bands were excised (white dots) and submitted to identification by MALDI-TOF mass fingerprint analysis. The proteins identified are given along with their positions. Abbreviations used: BSA, bovine serum albumin; BSF, bovine serum fetuin-A; b Apo-A1, bovine apolipoprotein A1; HSA, human serum albumin; HSF, human serum fetuin-A; and h Apo-A1, human apolipoprotein A1. Other minor protein bands include a bovine 95 kDa band (▪) which was identified as bovine serotransferrin precursor (2 out of 2 trials, or 2/2), and other minor human bands at 165 kDa (•), 130 kDa (♦), 95 kDa (◃) identified as human complement C3 (2/2), human antithrombin (2/2), and human complement C3 (2/2), respectively.

    Journal: PLoS ONE

    Article Title: Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    doi: 10.1371/journal.pone.0004417

    Figure Lengend Snippet: Protein identification in NLP prepared from FBS and HS. Protein profiles correspond to Figs. 11C and D , reproduced here to illustrate the protein identification procedure used as well as the results obtained. NLP were prepared from DMEM containing 5% FBS or 5% HS that had been precipitated with 10 mM each of CaCl 2 , Na 2 CO 3 , and NaH 2 PO 4 . Following SDS-PAGE and Coomassie blue staining, the bands were excised (white dots) and submitted to identification by MALDI-TOF mass fingerprint analysis. The proteins identified are given along with their positions. Abbreviations used: BSA, bovine serum albumin; BSF, bovine serum fetuin-A; b Apo-A1, bovine apolipoprotein A1; HSA, human serum albumin; HSF, human serum fetuin-A; and h Apo-A1, human apolipoprotein A1. Other minor protein bands include a bovine 95 kDa band (▪) which was identified as bovine serotransferrin precursor (2 out of 2 trials, or 2/2), and other minor human bands at 165 kDa (•), 130 kDa (♦), 95 kDa (◃) identified as human complement C3 (2/2), human antithrombin (2/2), and human complement C3 (2/2), respectively.

    Article Snippet: All strains of NB were cultured as described earlier , using DMEM with or without FBS (Biological Industries, Kibbutz Beit Haemek, Isreal ; PAA Laboratories, Pashing, Austria).

    Techniques: SDS Page, Staining

    Count of Vero cells cultured in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments ( P > 0.05).

    Journal: Blood research

    Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

    doi: 10.5045/br.2020.55.1.35

    Figure Lengend Snippet: Count of Vero cells cultured in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments ( P > 0.05).

    Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

    Techniques: Cell Culture

    Growth factor contents in FBS and HPL samples. IGF-1 (A) , TGF-β (B) , VEGF (C) , EGF (D) , FGF (E) , PDGF (F) . Values are expressed as means±SD (N=3). a) Significantly different from FBS samples at P

    Journal: Blood research

    Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

    doi: 10.5045/br.2020.55.1.35

    Figure Lengend Snippet: Growth factor contents in FBS and HPL samples. IGF-1 (A) , TGF-β (B) , VEGF (C) , EGF (D) , FGF (E) , PDGF (F) . Values are expressed as means±SD (N=3). a) Significantly different from FBS samples at P

    Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

    Techniques:

    Growth factor stability in HPL and FBS samples stored over 15 months. VEGF (A) , IGF-1 (B) , TGF-β (C) . Values are expressed as means±SD (N=3).

    Journal: Blood research

    Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

    doi: 10.5045/br.2020.55.1.35

    Figure Lengend Snippet: Growth factor stability in HPL and FBS samples stored over 15 months. VEGF (A) , IGF-1 (B) , TGF-β (C) . Values are expressed as means±SD (N=3).

    Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

    Techniques:

    (A) MTT reduction curve for Vero cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM. Data are expressed as means of three independent experiments ( P

    Journal: Blood research

    Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

    doi: 10.5045/br.2020.55.1.35

    Figure Lengend Snippet: (A) MTT reduction curve for Vero cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM. Data are expressed as means of three independent experiments ( P

    Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

    Techniques: MTT Assay, Cell Culture

    Count of Hep-2 cells cultivated in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments ( P > 0.05).

    Journal: Blood research

    Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

    doi: 10.5045/br.2020.55.1.35

    Figure Lengend Snippet: Count of Hep-2 cells cultivated in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments ( P > 0.05).

    Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

    Techniques:

    (A) MTT reduction curve for Hep-2 cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM ( P

    Journal: Blood research

    Article Title: Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

    doi: 10.5045/br.2020.55.1.35

    Figure Lengend Snippet: (A) MTT reduction curve for Hep-2 cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM ( P

    Article Snippet: Measurement of growth factors in HPL and FBS samples For HPL and FBS (#S1810-100, Biowest, Riverside, MO, USA) samples, the concentrations of IGF-1 and TGF-β1 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits #IGF31-K01 (Eagle Biosciences, Inc., Amherst, NH, USA) and #SK00033-02 (Aviscera Bioscience Inc., Santa Clara, CA, USA), respectively.

    Techniques: MTT Assay, Cell Culture

    In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Journal: Cancers

    Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

    doi: 10.3390/cancers12082085

    Figure Lengend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Article Snippet: Isolated pDCs and T lymphocytes (5 × 105 cells/mL) were cultured in RPMI 1640 medium (Biochrom GmbH, Holliston, MA, USA) with 10% FBS (Biochrom GmbH, Holliston, MA, USA), and 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to pDCs’ culture medium. pDCs and T lymphocytes were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), containing lactic acid (LA) (Sigma-Aldrich, St. Louis, MO, USA) or sodium lactate (NaL) (Alfa Aesar, Haverhill, MA, USA) at the concentrations of 10 mM, 15 mM, and 20 mM.

    Techniques: In Vitro, Purification, Cell Culture, Flow Cytometry

    Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Journal: Cancers

    Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

    doi: 10.3390/cancers12082085

    Figure Lengend Snippet: Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

    Article Snippet: Isolated pDCs and T lymphocytes (5 × 105 cells/mL) were cultured in RPMI 1640 medium (Biochrom GmbH, Holliston, MA, USA) with 10% FBS (Biochrom GmbH, Holliston, MA, USA), and 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to pDCs’ culture medium. pDCs and T lymphocytes were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), containing lactic acid (LA) (Sigma-Aldrich, St. Louis, MO, USA) or sodium lactate (NaL) (Alfa Aesar, Haverhill, MA, USA) at the concentrations of 10 mM, 15 mM, and 20 mM.

    Techniques: Purification, Cell Culture, Staining, Flow Cytometry

    Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

    Journal: Cancers

    Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

    doi: 10.3390/cancers12082085

    Figure Lengend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

    Article Snippet: Isolated pDCs and T lymphocytes (5 × 105 cells/mL) were cultured in RPMI 1640 medium (Biochrom GmbH, Holliston, MA, USA) with 10% FBS (Biochrom GmbH, Holliston, MA, USA), and 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to pDCs’ culture medium. pDCs and T lymphocytes were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), containing lactic acid (LA) (Sigma-Aldrich, St. Louis, MO, USA) or sodium lactate (NaL) (Alfa Aesar, Haverhill, MA, USA) at the concentrations of 10 mM, 15 mM, and 20 mM.

    Techniques: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

    Effect of incubates of platelets activated by collagen or TRAP-6 in the presence or absence of aspirin on HMEC-1 tube formation. HMEC-1 cells were resuspended in platelet incubates diluted with RPMI 1640 medium + 2% FBS and (1:1) and seeded (40,000 cells/well) on a 48-well plate coated with growth factor-reduced basement matrix extract. Platelet incubates contained human PRP stimulated with collagen (30 µg/ml) or TRAP-6 (30 µM) in the presence or absence of aspirin (100 µM) which were added to the cell suspensions. A ) Tube formation was visualized using time-lapse microscopy, where images were taken in phase every 30 min for 16 h (Cell M software). Images (original magnification, ×10) were taken after 16 h incubation (37°C; 5% CO 2 ) in phase or after the addition of calcein AM. B ) The number of branching points was quantitated after 16 h incubation using ImageJ software. Collagen-stimulated or TRAP-6-stimulated platelets significantly increased HMEC-1 tube formation. This effect was markedly reduced in response to aspirin-treated platelets. Data are shown as means ± sem (1-way ANOVA; n = 6). * P

    Journal: The FASEB Journal

    Article Title: Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1

    doi: 10.1096/fj.201600530R

    Figure Lengend Snippet: Effect of incubates of platelets activated by collagen or TRAP-6 in the presence or absence of aspirin on HMEC-1 tube formation. HMEC-1 cells were resuspended in platelet incubates diluted with RPMI 1640 medium + 2% FBS and (1:1) and seeded (40,000 cells/well) on a 48-well plate coated with growth factor-reduced basement matrix extract. Platelet incubates contained human PRP stimulated with collagen (30 µg/ml) or TRAP-6 (30 µM) in the presence or absence of aspirin (100 µM) which were added to the cell suspensions. A ) Tube formation was visualized using time-lapse microscopy, where images were taken in phase every 30 min for 16 h (Cell M software). Images (original magnification, ×10) were taken after 16 h incubation (37°C; 5% CO 2 ) in phase or after the addition of calcein AM. B ) The number of branching points was quantitated after 16 h incubation using ImageJ software. Collagen-stimulated or TRAP-6-stimulated platelets significantly increased HMEC-1 tube formation. This effect was markedly reduced in response to aspirin-treated platelets. Data are shown as means ± sem (1-way ANOVA; n = 6). * P

    Article Snippet: Rings were then incubated (37°C; 5% CO2 ) in Opti-MEM (Thermo Fisher Scientific) plus 1% FBS (PAA Laboratories/GE Healthcare, Little Chalfont, United Kingdom) in the presence of platelet releasates and/or VEGF (10 nM), 15(S )-HETE (1 µM), or vehicle.

    Techniques: Time-lapse Microscopy, Software, Incubation

    Effect of 15( S )-HETE on HMEC-1 tube formation in the presence of TRAP-6-stimulated platelet releasates. HMEC-1 cells were resuspended in platelet releasates diluted with RPMI 1640 medium + 2% FBS and (1:1) and seeded (40,000 cells/well) on a 48-well plate coated with growth factor-reduced basement matrix extract. Platelet releasates were obtained by centrifugation of human PRP stimulated with PBS or TRAP-6 (30 µM) in the presence or absence of aspirin (100 µM). The HMEC-1 cell suspensions were then treated with platelet releasates plus vehicle or 15( S )-HETE (1 µM). A ) Tube formation was visualized using time-lapse microscopy, where images were taken in phase every 30 min for 16 h (Cell M software). Images (original magnification, ×10) were taken after 16 h incubation (37°C; 5% CO 2 ) in phase or after the addition of calcein AM. B – D ) The number of branching points was quantitated after 4 h ( B ), 8 h ( C ), and 16 h ( D ) incubation using ImageJ software. TRAP-6-stimulated platelet releasates significantly increased HMEC-1 tube formation. This effect was markedly reduced in response to releasates prepared from aspirin-treated platelets, but fully restored by addition of 15( S )-HETE (1 µM). Data shown as means ± sem (1-way ANOVA; n = 3). * P

    Journal: The FASEB Journal

    Article Title: Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1

    doi: 10.1096/fj.201600530R

    Figure Lengend Snippet: Effect of 15( S )-HETE on HMEC-1 tube formation in the presence of TRAP-6-stimulated platelet releasates. HMEC-1 cells were resuspended in platelet releasates diluted with RPMI 1640 medium + 2% FBS and (1:1) and seeded (40,000 cells/well) on a 48-well plate coated with growth factor-reduced basement matrix extract. Platelet releasates were obtained by centrifugation of human PRP stimulated with PBS or TRAP-6 (30 µM) in the presence or absence of aspirin (100 µM). The HMEC-1 cell suspensions were then treated with platelet releasates plus vehicle or 15( S )-HETE (1 µM). A ) Tube formation was visualized using time-lapse microscopy, where images were taken in phase every 30 min for 16 h (Cell M software). Images (original magnification, ×10) were taken after 16 h incubation (37°C; 5% CO 2 ) in phase or after the addition of calcein AM. B – D ) The number of branching points was quantitated after 4 h ( B ), 8 h ( C ), and 16 h ( D ) incubation using ImageJ software. TRAP-6-stimulated platelet releasates significantly increased HMEC-1 tube formation. This effect was markedly reduced in response to releasates prepared from aspirin-treated platelets, but fully restored by addition of 15( S )-HETE (1 µM). Data shown as means ± sem (1-way ANOVA; n = 3). * P

    Article Snippet: Rings were then incubated (37°C; 5% CO2 ) in Opti-MEM (Thermo Fisher Scientific) plus 1% FBS (PAA Laboratories/GE Healthcare, Little Chalfont, United Kingdom) in the presence of platelet releasates and/or VEGF (10 nM), 15(S )-HETE (1 µM), or vehicle.

    Techniques: Centrifugation, Time-lapse Microscopy, Software, Incubation

    Effect of 15( S )-HETE on formation of angiogenic sprouts from rat aortic rings in the presence of collagen or TRAP-6-stimulated platelet releasates. Platelet releasates prepared as in Fig. 5 were diluted in Opti-MEM + 1% FBS (1:3) with the addition of supplemental collagen or TRAP-6 and/or aspirin to provide common incubation solutions for all rings. Rings incubated with PBS were treated without ( A , H ) or with ( B , I ) VEGF (10 ng/ml) or 15( S )-HETE (1 µM) ( C , J ), whereas rings incubated with releasates from platelets treated with collagen ( D – F ) or TRAP-6 ( K – M ) were treated without or with 15( S )-HETE (1 µM). Once additions had been made, aortic rings (5/condition) were incubated (37°C, 5% CO 2 ) for 5 d. Images were taken in phase (original magnification, ×4). G , N ) The number of sprouts was determined using Cell Ml software. A , H ) PBS alone did not stimulate formation of sprouts. B , C , I , J ) In contrast, the presence of VEGF significantly increased the number of sprouts ( B , I ), whereas 15( S )-HETE produced a moderate increase ( C , J ). D , E , K , L ) Releasates from platelets stimulated with collagen ( D ) or TRAP-6 ( K ) significantly promoted the formation of sprouts, whereas releasates from platelets treated with aspirin had much weaker effects ( E , L ). F , M ) The addition of 15( S )-HETE (1 µM) to aspirin-treated platelet releasates restored the formation of angiogenic sprouts. One-sample test used to compare differences to control response (PBS + VEGF; n = 3 for each). * P

    Journal: The FASEB Journal

    Article Title: Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1

    doi: 10.1096/fj.201600530R

    Figure Lengend Snippet: Effect of 15( S )-HETE on formation of angiogenic sprouts from rat aortic rings in the presence of collagen or TRAP-6-stimulated platelet releasates. Platelet releasates prepared as in Fig. 5 were diluted in Opti-MEM + 1% FBS (1:3) with the addition of supplemental collagen or TRAP-6 and/or aspirin to provide common incubation solutions for all rings. Rings incubated with PBS were treated without ( A , H ) or with ( B , I ) VEGF (10 ng/ml) or 15( S )-HETE (1 µM) ( C , J ), whereas rings incubated with releasates from platelets treated with collagen ( D – F ) or TRAP-6 ( K – M ) were treated without or with 15( S )-HETE (1 µM). Once additions had been made, aortic rings (5/condition) were incubated (37°C, 5% CO 2 ) for 5 d. Images were taken in phase (original magnification, ×4). G , N ) The number of sprouts was determined using Cell Ml software. A , H ) PBS alone did not stimulate formation of sprouts. B , C , I , J ) In contrast, the presence of VEGF significantly increased the number of sprouts ( B , I ), whereas 15( S )-HETE produced a moderate increase ( C , J ). D , E , K , L ) Releasates from platelets stimulated with collagen ( D ) or TRAP-6 ( K ) significantly promoted the formation of sprouts, whereas releasates from platelets treated with aspirin had much weaker effects ( E , L ). F , M ) The addition of 15( S )-HETE (1 µM) to aspirin-treated platelet releasates restored the formation of angiogenic sprouts. One-sample test used to compare differences to control response (PBS + VEGF; n = 3 for each). * P

    Article Snippet: Rings were then incubated (37°C; 5% CO2 ) in Opti-MEM (Thermo Fisher Scientific) plus 1% FBS (PAA Laboratories/GE Healthcare, Little Chalfont, United Kingdom) in the presence of platelet releasates and/or VEGF (10 nM), 15(S )-HETE (1 µM), or vehicle.

    Techniques: Incubation, Software, Produced

    Uptake of B. anthracis spores into mammalian cells cultured under germinating or non-germinating conditions . RAW264.7 cells (A, D), MH-S cells (B, E), or JAWSII cells (C, F) were incubated with B. anthracis spores (MOI 10) in DMEM, RPMI, or DMEM, respectively, in the presence (+, black bars) or absence (-, white bars) of FBS (10%), and then evaluated at 5 or 60 min by flow cytometry and in the presence of trypan blue (0.5%) for the percentage of cells with intracellular spores (A-C), and, for total cell associated spore fluorescence (D-F), as described under Materials and Methods. (A-C) The data are rendered as the percentage of infected cells with the entire population that has internalized spores. (D-F) The data are expressed as the change in MFI, normalized to cells at 5 min post infection in FBS-free medium. To generate the bar graphs, data were combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences in percent infected cells (A) or total intracellular spores (B) between cells incubated in the absence or presence of FBS.

    Journal: BMC Microbiology

    Article Title: Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    doi: 10.1186/1471-2180-11-46

    Figure Lengend Snippet: Uptake of B. anthracis spores into mammalian cells cultured under germinating or non-germinating conditions . RAW264.7 cells (A, D), MH-S cells (B, E), or JAWSII cells (C, F) were incubated with B. anthracis spores (MOI 10) in DMEM, RPMI, or DMEM, respectively, in the presence (+, black bars) or absence (-, white bars) of FBS (10%), and then evaluated at 5 or 60 min by flow cytometry and in the presence of trypan blue (0.5%) for the percentage of cells with intracellular spores (A-C), and, for total cell associated spore fluorescence (D-F), as described under Materials and Methods. (A-C) The data are rendered as the percentage of infected cells with the entire population that has internalized spores. (D-F) The data are expressed as the change in MFI, normalized to cells at 5 min post infection in FBS-free medium. To generate the bar graphs, data were combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences in percent infected cells (A) or total intracellular spores (B) between cells incubated in the absence or presence of FBS.

    Article Snippet: In other studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM (Mediatech), BME (Sigma), CIM (Gibco), Ham's F-12 (Mediatech), McCoy's 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma).

    Techniques: Cell Culture, Incubation, Flow Cytometry, Cytometry, Fluorescence, Infection

    The germination state of spores influences the viability of B. anthracis -infected cells . RAW264.7 cells were incubated for 30 min with B. anthracis spores (MOI 10) in DMEM in the presence (+, black bars) or absence (-, white bars) of FBS (10%). After 30 min, the cells were washed to remove extracellular B. anthracis , and then further incubated with FBS (10%) and, as described under

    Journal: BMC Microbiology

    Article Title: Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    doi: 10.1186/1471-2180-11-46

    Figure Lengend Snippet: The germination state of spores influences the viability of B. anthracis -infected cells . RAW264.7 cells were incubated for 30 min with B. anthracis spores (MOI 10) in DMEM in the presence (+, black bars) or absence (-, white bars) of FBS (10%). After 30 min, the cells were washed to remove extracellular B. anthracis , and then further incubated with FBS (10%) and, as described under "Methods," with gentamicin to germinate and kill any remaining spores that had not been germinated. After 15 min, the cells were washed and then further incubated in the absence of gentamicin. At 0 (immediately after gentamicin removal), 60, or 240 min after removal of gentamicin, as indicated, the cells were evaluated for mammalian cell death via PI uptake, as described under Materials and Methods. The data are rendered as the fold-increase of PI uptake relative to non-infected cells in the absence or presence of FBS at 5, 60, or 240 min, as indicated. The rendered data have been combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences between the fold-increase of PI uptake between cells incubated with spores in the absence or presence of FBS.

    Article Snippet: In other studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM (Mediatech), BME (Sigma), CIM (Gibco), Ham's F-12 (Mediatech), McCoy's 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma).

    Techniques: Infection, Incubation

    The effects of pre-conditioned culture medium on the germination state of B. anthracis spores . DMEM (A, B) or RPMI (B) were pre-conditioned by incubating with monolayers of RAW264.7 (A, B) or MH-S cells (B) at 37°C and under 5% CO 2 , in the absence (A) or presence (MOI 10) (B) of B. anthracis spores. (A, B). After 4 h (white bars) or 24 h (black bars) (A), or after 1 (white bars) and 4 h (black bars) (B), the medium was removed from the monolayers, filter sterilized, and then incubated with B. anthracis spores in 96-well plates at 37°C and with rotary agitation. Germination and outgrowth of spores were monitored at indicated times by measuring O.D. 600 nm . The results are rendered as the O.D. 600 nm of the spore suspension at the indicated time relative to the original O.D. 600 nm of the spore suspension at time = 0 of the 37°C incubation. P -values were calculated to evaluate the statistical significance of the differences between O.D. 600 nm values at the initial time point and O.D. O.D. 600 nm values at the indicated times. For (B), BHI (gray bars) was used as a positive control for germination and outgrowth. (C) An equal number of B. anthracis spores were incubated at 37°C and under 5% CO 2 in DMEM (no FBS) in the absence (white bars) or presence (black bars) of RAW264.7 cells (MOI 10). At indicated times, aliquots of culture medium were removed, and spores were evaluated for heat resistance. The results are rendered as the number of heat resistant spores relative to spores incubated in DMEM alone, which were normalized to 1.0. P -values were calculated to evaluate the statistical significance of the differences in heat resistant spores between those incubated in the presence or absence of RAW264.7 cells. The data in (A-C) are combined from 3 independent experiments conducted in triplicate with error bars indicating standard deviations.

    Journal: BMC Microbiology

    Article Title: Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    doi: 10.1186/1471-2180-11-46

    Figure Lengend Snippet: The effects of pre-conditioned culture medium on the germination state of B. anthracis spores . DMEM (A, B) or RPMI (B) were pre-conditioned by incubating with monolayers of RAW264.7 (A, B) or MH-S cells (B) at 37°C and under 5% CO 2 , in the absence (A) or presence (MOI 10) (B) of B. anthracis spores. (A, B). After 4 h (white bars) or 24 h (black bars) (A), or after 1 (white bars) and 4 h (black bars) (B), the medium was removed from the monolayers, filter sterilized, and then incubated with B. anthracis spores in 96-well plates at 37°C and with rotary agitation. Germination and outgrowth of spores were monitored at indicated times by measuring O.D. 600 nm . The results are rendered as the O.D. 600 nm of the spore suspension at the indicated time relative to the original O.D. 600 nm of the spore suspension at time = 0 of the 37°C incubation. P -values were calculated to evaluate the statistical significance of the differences between O.D. 600 nm values at the initial time point and O.D. O.D. 600 nm values at the indicated times. For (B), BHI (gray bars) was used as a positive control for germination and outgrowth. (C) An equal number of B. anthracis spores were incubated at 37°C and under 5% CO 2 in DMEM (no FBS) in the absence (white bars) or presence (black bars) of RAW264.7 cells (MOI 10). At indicated times, aliquots of culture medium were removed, and spores were evaluated for heat resistance. The results are rendered as the number of heat resistant spores relative to spores incubated in DMEM alone, which were normalized to 1.0. P -values were calculated to evaluate the statistical significance of the differences in heat resistant spores between those incubated in the presence or absence of RAW264.7 cells. The data in (A-C) are combined from 3 independent experiments conducted in triplicate with error bars indicating standard deviations.

    Article Snippet: In other studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM (Mediatech), BME (Sigma), CIM (Gibco), Ham's F-12 (Mediatech), McCoy's 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma).

    Techniques: Incubation, Positive Control

    Effect of non-germinating conditions on RAW264.7 cell viability, cell cycle progression, and metabolic activity . RAW264.7 cells were incubated at 37° in DMEM in the presence (+, black bars) or absence (-, white bars) of FBS, and then evaluated at 4 or 24 h, as indicated, for viability (A), cell cycle progression (B), and metabolic activity (C). (A) The cells were assayed for PI uptake, as described under Materials and Methods. The data are rendered as the relative PI uptake normalized at both 4 and 24 h to cells incubated in the absence of FBS. (B) The cells were analyzed for their cell cycle profiles, as described under Materials and Methods. The data are rendered as the relative numbers of cells in G 2 /M normalized at both 4 and 24 h to cells incubated in the absence of FBS. (C) The cells were analyzed for conversion of MTT to formazan. The data are rendered as the fold change of formazan production normalized at both 4 and 24 h to cells incubated in the absence of FBS. To generate the bar graphs, data were combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences in viability (A), cell cycle progression (B), and metabolism (C) between cells cultured in the absence or presence of FBS.

    Journal: BMC Microbiology

    Article Title: Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    doi: 10.1186/1471-2180-11-46

    Figure Lengend Snippet: Effect of non-germinating conditions on RAW264.7 cell viability, cell cycle progression, and metabolic activity . RAW264.7 cells were incubated at 37° in DMEM in the presence (+, black bars) or absence (-, white bars) of FBS, and then evaluated at 4 or 24 h, as indicated, for viability (A), cell cycle progression (B), and metabolic activity (C). (A) The cells were assayed for PI uptake, as described under Materials and Methods. The data are rendered as the relative PI uptake normalized at both 4 and 24 h to cells incubated in the absence of FBS. (B) The cells were analyzed for their cell cycle profiles, as described under Materials and Methods. The data are rendered as the relative numbers of cells in G 2 /M normalized at both 4 and 24 h to cells incubated in the absence of FBS. (C) The cells were analyzed for conversion of MTT to formazan. The data are rendered as the fold change of formazan production normalized at both 4 and 24 h to cells incubated in the absence of FBS. To generate the bar graphs, data were combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences in viability (A), cell cycle progression (B), and metabolism (C) between cells cultured in the absence or presence of FBS.

    Article Snippet: In other studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM (Mediatech), BME (Sigma), CIM (Gibco), Ham's F-12 (Mediatech), McCoy's 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma).

    Techniques: Activity Assay, Incubation, MTT Assay, Cell Culture

    The germination state of spores influences the viability of intracellular B. anthracis . RAW264.7 cells were incubated for 30 min with dormant B. anthracis spores (MOI 10) in DMEM in the presence (+, black bars) or absence (-, white bars) of FBS (10%), or, with pre-germinated spores (MOI 10) in DMEM in the absence of FBS (grey bars). B. anthracis spores were pre-germinated by incubation for 30 min in PBS pH 7.2 supplemented with L-alanine and L-inosine (both at 10 mM), and then washed twice with PBS pH 7.2 to remove germinants. After 30 min, the cells were washed to remove extracellular B. anthracis , and then further incubated with FBS (10%) and, as described under

    Journal: BMC Microbiology

    Article Title: Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    doi: 10.1186/1471-2180-11-46

    Figure Lengend Snippet: The germination state of spores influences the viability of intracellular B. anthracis . RAW264.7 cells were incubated for 30 min with dormant B. anthracis spores (MOI 10) in DMEM in the presence (+, black bars) or absence (-, white bars) of FBS (10%), or, with pre-germinated spores (MOI 10) in DMEM in the absence of FBS (grey bars). B. anthracis spores were pre-germinated by incubation for 30 min in PBS pH 7.2 supplemented with L-alanine and L-inosine (both at 10 mM), and then washed twice with PBS pH 7.2 to remove germinants. After 30 min, the cells were washed to remove extracellular B. anthracis , and then further incubated with FBS (10%) and, as described under "Methods," with gentamicin to germinate and kill any remaining spores that had not been germinated. After 15 min, the cells were washed and then further incubated in the absence of gentamicin. At 5, 60, or 240 min after removal of gentamicin, as indicated, the RAW264.7 cells were lysed, and the lysates were evaluated for viable B. anthracis , as described under Materials and Methods. The data were rendered as the fold-change in recoverable CFU in the absence or presence of FBS, relative to cells at 5 min post infection in the absence of FBS. The rendered data were combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences in recoverable CFU between cells infected in the absence or presence of FBS.

    Article Snippet: In other studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM (Mediatech), BME (Sigma), CIM (Gibco), Ham's F-12 (Mediatech), McCoy's 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma).

    Techniques: Incubation, Infection

    FBS in cell culture media promotes germination and outgrowth of B. anthracis spores . B. anthracis spores were incubated in 96-well plates at 37°C and with rotary agitation in the indicated medium. Germination and outgrowth of spores were monitored at the indicated times. Medium conditions are listed at the top of the figure, and applicable to (A-C). (A) Optical determination of germination and outgrowth. The data are rendered as the O.D. 600 nm of the spore suspension at the indicated times relative to the original O.D. 600 nm of the spore suspension at time = 0 of the 37°C incubation. For BHI, DMEM, RPMI, and MEMα, initial decreases in O.D. 600 nm reflect the loss of spore refractility that occurs subsequent to germination initiation, while the increases in O.D. 600 nm measured at later time points (1 and 4 h) reflects bacterial replication. For PBS, the modest increases in O.D. 600 nm are due to time-dependent medium evaporation. Error bars indicate standard deviations. For each medium tested, the P -values were calculated to evaluate the statistical significance of the differences between O.D. 600 nm values at the indicated times and O.D. 600 nm values at the initial time point. (B) Spore heat sensitivity as a function of medium conditions. Aliquots from spore cultures were removed at indicated times, incubated for 30 min at either at 65°C or on ice, diluted 10 1 - or 10 2 -fold (PBS pH 7.2), spotted (10 μL) on LB plates, and incubated at 25°C. After 18 h, the plates were photographed. (C) Visual determination of B. anthracis spore outgrowth as a function of cell culture medium. Aliquots from spore cultures were removed at indicated times and analyzed for outgrowth using DIC microscopy. The bars indicate a length of 6.5 μm. The data in (A) are combined from 3 independent experiments. The data in (B) and (C) are from a single experiment, and are representative of 3 independent experiments.

    Journal: BMC Microbiology

    Article Title: Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

    doi: 10.1186/1471-2180-11-46

    Figure Lengend Snippet: FBS in cell culture media promotes germination and outgrowth of B. anthracis spores . B. anthracis spores were incubated in 96-well plates at 37°C and with rotary agitation in the indicated medium. Germination and outgrowth of spores were monitored at the indicated times. Medium conditions are listed at the top of the figure, and applicable to (A-C). (A) Optical determination of germination and outgrowth. The data are rendered as the O.D. 600 nm of the spore suspension at the indicated times relative to the original O.D. 600 nm of the spore suspension at time = 0 of the 37°C incubation. For BHI, DMEM, RPMI, and MEMα, initial decreases in O.D. 600 nm reflect the loss of spore refractility that occurs subsequent to germination initiation, while the increases in O.D. 600 nm measured at later time points (1 and 4 h) reflects bacterial replication. For PBS, the modest increases in O.D. 600 nm are due to time-dependent medium evaporation. Error bars indicate standard deviations. For each medium tested, the P -values were calculated to evaluate the statistical significance of the differences between O.D. 600 nm values at the indicated times and O.D. 600 nm values at the initial time point. (B) Spore heat sensitivity as a function of medium conditions. Aliquots from spore cultures were removed at indicated times, incubated for 30 min at either at 65°C or on ice, diluted 10 1 - or 10 2 -fold (PBS pH 7.2), spotted (10 μL) on LB plates, and incubated at 25°C. After 18 h, the plates were photographed. (C) Visual determination of B. anthracis spore outgrowth as a function of cell culture medium. Aliquots from spore cultures were removed at indicated times and analyzed for outgrowth using DIC microscopy. The bars indicate a length of 6.5 μm. The data in (A) are combined from 3 independent experiments. The data in (B) and (C) are from a single experiment, and are representative of 3 independent experiments.

    Article Snippet: In other studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM (Mediatech), BME (Sigma), CIM (Gibco), Ham's F-12 (Mediatech), McCoy's 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma).

    Techniques: Cell Culture, Incubation, Evaporation, Microscopy

    Effect of insulin-like growth factor (IGF)-1 on alkaline phosphatase activity in osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 nM 1,25(OH) 2 D 3 in Ham F12/DMEM media containing 2% charcoal-treated FBS and in the presence or not of 50 ng/ml IGF-1 with or without 10 μM PD98059 (PD). Results are expressed as the mean ± SEM of control values without IGF-1 for 6 OA osteoblast preparations.

    Journal: Arthritis Research & Therapy

    Article Title: Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts

    doi: 10.1186/ar2087

    Figure Lengend Snippet: Effect of insulin-like growth factor (IGF)-1 on alkaline phosphatase activity in osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 nM 1,25(OH) 2 D 3 in Ham F12/DMEM media containing 2% charcoal-treated FBS and in the presence or not of 50 ng/ml IGF-1 with or without 10 μM PD98059 (PD). Results are expressed as the mean ± SEM of control values without IGF-1 for 6 OA osteoblast preparations.

    Article Snippet: At confluence, cells were passaged once at 25,000 cells/cm2 in 6 well plates and grown for 5 days in Ham F12/DMEM (Sigma, St-Louis, MO, USA) containing 10% FBS (Wisent Inc., St Bruno, Quebec, Canada) before specific assays.

    Techniques: Activity Assay, Incubation

    Cell proliferation of normal and osteoarthitis (OA) osteoblasts in response to insulin-like growth factor (IGF)-1 stimulation. Cells were plated at 10,000 cells/cm 2 in 96-well plates and incubated overnight in Ham F12/DMEM media containing 10% FBS. Cells were then serum-starved for 24 hours in the same media containing 0.5% BSA then treated with or without 50 ng/ml IGF-1 in the presence or not of 10 μM PD98059 (PD) for 24 hours followed by incubation with bromodeoxyuridine (BrdU) for their last 24 hours of incubation in the same media. BrdU incorporation was then evaluated following the manufacturer's instructions. Results are expressed as mean OD units ± SEM for three normal and four OA osteoblast preparations.

    Journal: Arthritis Research & Therapy

    Article Title: Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts

    doi: 10.1186/ar2087

    Figure Lengend Snippet: Cell proliferation of normal and osteoarthitis (OA) osteoblasts in response to insulin-like growth factor (IGF)-1 stimulation. Cells were plated at 10,000 cells/cm 2 in 96-well plates and incubated overnight in Ham F12/DMEM media containing 10% FBS. Cells were then serum-starved for 24 hours in the same media containing 0.5% BSA then treated with or without 50 ng/ml IGF-1 in the presence or not of 10 μM PD98059 (PD) for 24 hours followed by incubation with bromodeoxyuridine (BrdU) for their last 24 hours of incubation in the same media. BrdU incorporation was then evaluated following the manufacturer's instructions. Results are expressed as mean OD units ± SEM for three normal and four OA osteoblast preparations.

    Article Snippet: At confluence, cells were passaged once at 25,000 cells/cm2 in 6 well plates and grown for 5 days in Ham F12/DMEM (Sigma, St-Louis, MO, USA) containing 10% FBS (Wisent Inc., St Bruno, Quebec, Canada) before specific assays.

    Techniques: Incubation, BrdU Incorporation Assay

    Proliferation kinetics of UCMSCs in CBS and FBS supplemented media. Initial densities at each passage were 10,000/well and results are shown as means±SE. (A) Population doublings time (PDT). (B) Fold increase. *p-value

    Journal: International Journal of Stem Cells

    Article Title: A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum

    doi: 10.15283/ijsc17028

    Figure Lengend Snippet: Proliferation kinetics of UCMSCs in CBS and FBS supplemented media. Initial densities at each passage were 10,000/well and results are shown as means±SE. (A) Population doublings time (PDT). (B) Fold increase. *p-value

    Article Snippet: The segments were cut longitudinally, and blood vessels were removed, and segments transferred to 25 cm2 flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Euroclone, Italy) supplemented with either 10% FBS (Euroclone, Italy) or 10% CBS.

    Techniques:

    Fibroblastic morphology of MSCs isolated from 5 cm 2 umbilical cord explants and expanded in FBS supplement medium (A~C) or CBS supplement medium (D~F). (A, D) migrating cells appearing at the edge of explants after 7 days (150×). (B, E) adherent cells after removing explants (600×). (C, F) MSCs at confluence in passage 3 (300×).

    Journal: International Journal of Stem Cells

    Article Title: A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum

    doi: 10.15283/ijsc17028

    Figure Lengend Snippet: Fibroblastic morphology of MSCs isolated from 5 cm 2 umbilical cord explants and expanded in FBS supplement medium (A~C) or CBS supplement medium (D~F). (A, D) migrating cells appearing at the edge of explants after 7 days (150×). (B, E) adherent cells after removing explants (600×). (C, F) MSCs at confluence in passage 3 (300×).

    Article Snippet: The segments were cut longitudinally, and blood vessels were removed, and segments transferred to 25 cm2 flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Euroclone, Italy) supplemented with either 10% FBS (Euroclone, Italy) or 10% CBS.

    Techniques: Isolation

    2% agarose gel electrophoresis for amplified transcripts expressed by MSCs cultured in FBS supplement medium (A) or in CBS supplement medium (B). ( 1 , 5 ) beta-actin amplicon of 100bp used as control. ( 2 , 6 ) CD105 amplicon of 165 bp. (3. 7) CD90 amplicon of 124 bp. ( 4 , 8 ) CD44 amplicon of 233 bp.

    Journal: International Journal of Stem Cells

    Article Title: A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum

    doi: 10.15283/ijsc17028

    Figure Lengend Snippet: 2% agarose gel electrophoresis for amplified transcripts expressed by MSCs cultured in FBS supplement medium (A) or in CBS supplement medium (B). ( 1 , 5 ) beta-actin amplicon of 100bp used as control. ( 2 , 6 ) CD105 amplicon of 165 bp. (3. 7) CD90 amplicon of 124 bp. ( 4 , 8 ) CD44 amplicon of 233 bp.

    Article Snippet: The segments were cut longitudinally, and blood vessels were removed, and segments transferred to 25 cm2 flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Euroclone, Italy) supplemented with either 10% FBS (Euroclone, Italy) or 10% CBS.

    Techniques: Agarose Gel Electrophoresis, Amplification, Cell Culture

    Flow cytometry of UCMSCs cultured in human CBS supplemented medium (A~F) and in FBS supplemented medium (G~L). (A, G) FSC and SSC distribution of gated cells. Cells stained positive for CD90 (B, H), CD105 (C, I), CD44 (D, J), while stained negative for CD45 (E, K), and CD34 (F, L).

    Journal: International Journal of Stem Cells

    Article Title: A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum

    doi: 10.15283/ijsc17028

    Figure Lengend Snippet: Flow cytometry of UCMSCs cultured in human CBS supplemented medium (A~F) and in FBS supplemented medium (G~L). (A, G) FSC and SSC distribution of gated cells. Cells stained positive for CD90 (B, H), CD105 (C, I), CD44 (D, J), while stained negative for CD45 (E, K), and CD34 (F, L).

    Article Snippet: The segments were cut longitudinally, and blood vessels were removed, and segments transferred to 25 cm2 flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Euroclone, Italy) supplemented with either 10% FBS (Euroclone, Italy) or 10% CBS.

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Staining

    Immunofluorescence for CD105, CD44 and CD90 expression in MSCs cultured in CBS supplement media (A~C) or in FBS supplement media (D~F). Positive staining for CD105 (A, D), CD44 (B, E), and CD90 (C, F). Nuclei were counterstained with DAPI (blue).

    Journal: International Journal of Stem Cells

    Article Title: A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum

    doi: 10.15283/ijsc17028

    Figure Lengend Snippet: Immunofluorescence for CD105, CD44 and CD90 expression in MSCs cultured in CBS supplement media (A~C) or in FBS supplement media (D~F). Positive staining for CD105 (A, D), CD44 (B, E), and CD90 (C, F). Nuclei were counterstained with DAPI (blue).

    Article Snippet: The segments were cut longitudinally, and blood vessels were removed, and segments transferred to 25 cm2 flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Euroclone, Italy) supplemented with either 10% FBS (Euroclone, Italy) or 10% CBS.

    Techniques: Immunofluorescence, Expressing, Cell Culture, Staining

    Adipogenic and osteogenic differentiation of MSCs. (A, C, E, G) are controls were MSCs from passage 3 were cultured and stained similarly in the presence of FBS supplemented medium (A, E) or CBS supplemented medium (C, G). (B, D) osteogenic differentiation showed positive staining of mineralized by Alizarin red. (F, H) adipogenic differentiation showed positive staining of lipid vacuoles by Sudan III.

    Journal: International Journal of Stem Cells

    Article Title: A Simple Method to Isolate and Expand Human Umbilical Cord Derived Mesenchymal Stem Cells: Using Explant Method and Umbilical Cord Blood Serum

    doi: 10.15283/ijsc17028

    Figure Lengend Snippet: Adipogenic and osteogenic differentiation of MSCs. (A, C, E, G) are controls were MSCs from passage 3 were cultured and stained similarly in the presence of FBS supplemented medium (A, E) or CBS supplemented medium (C, G). (B, D) osteogenic differentiation showed positive staining of mineralized by Alizarin red. (F, H) adipogenic differentiation showed positive staining of lipid vacuoles by Sudan III.

    Article Snippet: The segments were cut longitudinally, and blood vessels were removed, and segments transferred to 25 cm2 flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Euroclone, Italy) supplemented with either 10% FBS (Euroclone, Italy) or 10% CBS.

    Techniques: Cell Culture, Staining

    Lipid raft and cholesterol content in T cells cultured in normal or delipidated medium. T cells isolated from naïve apoE-/- mice were cultured in culture medium supplemented with 10% delipidated FBS or 10% FBS medium for 24 hours with CD3/CD28 activation beads. More unesterified cholesterol contents (A) or lipid rafts (B) were observed in activated CD4 + and CD8 + T cells cultured in 10% FBS medium when compared to T cells cultured in 10% delipidated FBS medium. Experiments were repeated 4 times using T cells from 3 mice.

    Journal: PLoS ONE

    Article Title: Cholesterol Lowering Modulates T Cell Function In Vivo and In Vitro

    doi: 10.1371/journal.pone.0092095

    Figure Lengend Snippet: Lipid raft and cholesterol content in T cells cultured in normal or delipidated medium. T cells isolated from naïve apoE-/- mice were cultured in culture medium supplemented with 10% delipidated FBS or 10% FBS medium for 24 hours with CD3/CD28 activation beads. More unesterified cholesterol contents (A) or lipid rafts (B) were observed in activated CD4 + and CD8 + T cells cultured in 10% FBS medium when compared to T cells cultured in 10% delipidated FBS medium. Experiments were repeated 4 times using T cells from 3 mice.

    Article Snippet: Isolated T cells were loaded with 2.5 μM CFSE (Invitrogen) for 10 min and then seeded at 0.5×106 cells in 1 ml RPMI 1640 medium (invitrogen) supplemented with 10% FBS (Omega Scientific) or 10% delipidated FBS (Cocalico Biologicals Inc.), 1X Antibiotic-Antimycotic (Gibco) and 50 μM β-ME in 12 well plates.

    Techniques: Cell Culture, Isolation, Mouse Assay, Activation Assay

    T cell proliferation in normal or delipidated meidum using serum of the same lot. (A) T cells isolated from naïve apoE-/- mice proliferated less when cultured in medium supplemented with 10% delipidated FBS when compared to those cutured in medium supplemented with 10% FBS of the same lot. N = 4. (B) T cells isolated form naïve wild type mice proliferated less when cultured in medium supplemented with 10% delipidated FBS when compared to those cutured in medium supplemented with 10% FBS of the same lot. N = 4.

    Journal: PLoS ONE

    Article Title: Cholesterol Lowering Modulates T Cell Function In Vivo and In Vitro

    doi: 10.1371/journal.pone.0092095

    Figure Lengend Snippet: T cell proliferation in normal or delipidated meidum using serum of the same lot. (A) T cells isolated from naïve apoE-/- mice proliferated less when cultured in medium supplemented with 10% delipidated FBS when compared to those cutured in medium supplemented with 10% FBS of the same lot. N = 4. (B) T cells isolated form naïve wild type mice proliferated less when cultured in medium supplemented with 10% delipidated FBS when compared to those cutured in medium supplemented with 10% FBS of the same lot. N = 4.

    Article Snippet: Isolated T cells were loaded with 2.5 μM CFSE (Invitrogen) for 10 min and then seeded at 0.5×106 cells in 1 ml RPMI 1640 medium (invitrogen) supplemented with 10% FBS (Omega Scientific) or 10% delipidated FBS (Cocalico Biologicals Inc.), 1X Antibiotic-Antimycotic (Gibco) and 50 μM β-ME in 12 well plates.

    Techniques: Isolation, Mouse Assay, Cell Culture

    Western blot for pZap70 and IL-10 ELISA. (A) T cells isolated from naïve apoE-/- mice were cultured in 10% delipidated FBS medium or 10% FBS medium for 2 min with CD3/CD28 activation beads. Western blot analysis of the whole cell lysates revealed a higher expression of pZap-70 from T cells cultured in NM (medium supplemented with 10% FBS) when compared to that from T cells cultured in DM (medium supplemented with 10% delipidated FBS). Upper panel showed a representative Western blot and lower panel is the densitometric analysis from three experiments. N = 3 in each group. (B) IL-10 level was lower in delipidated medium of T cells after activation for 4 days with CD3/CD28 beads when compared to that in normal medium. N = 4.

    Journal: PLoS ONE

    Article Title: Cholesterol Lowering Modulates T Cell Function In Vivo and In Vitro

    doi: 10.1371/journal.pone.0092095

    Figure Lengend Snippet: Western blot for pZap70 and IL-10 ELISA. (A) T cells isolated from naïve apoE-/- mice were cultured in 10% delipidated FBS medium or 10% FBS medium for 2 min with CD3/CD28 activation beads. Western blot analysis of the whole cell lysates revealed a higher expression of pZap-70 from T cells cultured in NM (medium supplemented with 10% FBS) when compared to that from T cells cultured in DM (medium supplemented with 10% delipidated FBS). Upper panel showed a representative Western blot and lower panel is the densitometric analysis from three experiments. N = 3 in each group. (B) IL-10 level was lower in delipidated medium of T cells after activation for 4 days with CD3/CD28 beads when compared to that in normal medium. N = 4.

    Article Snippet: Isolated T cells were loaded with 2.5 μM CFSE (Invitrogen) for 10 min and then seeded at 0.5×106 cells in 1 ml RPMI 1640 medium (invitrogen) supplemented with 10% FBS (Omega Scientific) or 10% delipidated FBS (Cocalico Biologicals Inc.), 1X Antibiotic-Antimycotic (Gibco) and 50 μM β-ME in 12 well plates.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Mouse Assay, Cell Culture, Activation Assay, Expressing

    T cell proliferation in normal or delipidated medium. (A) T cells isolated from human peripheral blood were loaded with CFSE and cultured in culture medium supplemented with 10% delipidated FBS (delipidated) or 10% FBS (normal) for 4 days with CD3/CD28 activation beads. After activation, CD4 + and CD8 + T cells proliferated less when cultured in 10% delipidated FBS medium. Experiments were done in duplicates from the first sample, triplicates from the second sample and quadruplicates from the third sample using peripheral blood from 3 different human samples, hence n = 9 in each group. (B) Similarly CD4 + or CD8 + T cells isolated from naïve apoE-/- mice proliferated less when cultured in medium supplemented with 10% delipidated FBS when compared to those cultured in medium supplemented with 10% FBS. N = 6 in each group of CD4 + cells and n = 4 in each group of CD8 + cells.

    Journal: PLoS ONE

    Article Title: Cholesterol Lowering Modulates T Cell Function In Vivo and In Vitro

    doi: 10.1371/journal.pone.0092095

    Figure Lengend Snippet: T cell proliferation in normal or delipidated medium. (A) T cells isolated from human peripheral blood were loaded with CFSE and cultured in culture medium supplemented with 10% delipidated FBS (delipidated) or 10% FBS (normal) for 4 days with CD3/CD28 activation beads. After activation, CD4 + and CD8 + T cells proliferated less when cultured in 10% delipidated FBS medium. Experiments were done in duplicates from the first sample, triplicates from the second sample and quadruplicates from the third sample using peripheral blood from 3 different human samples, hence n = 9 in each group. (B) Similarly CD4 + or CD8 + T cells isolated from naïve apoE-/- mice proliferated less when cultured in medium supplemented with 10% delipidated FBS when compared to those cultured in medium supplemented with 10% FBS. N = 6 in each group of CD4 + cells and n = 4 in each group of CD8 + cells.

    Article Snippet: Isolated T cells were loaded with 2.5 μM CFSE (Invitrogen) for 10 min and then seeded at 0.5×106 cells in 1 ml RPMI 1640 medium (invitrogen) supplemented with 10% FBS (Omega Scientific) or 10% delipidated FBS (Cocalico Biologicals Inc.), 1X Antibiotic-Antimycotic (Gibco) and 50 μM β-ME in 12 well plates.

    Techniques: Isolation, Cell Culture, Activation Assay, Mouse Assay