trypsin edta  (Millipore)


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  • 99
    Name:
    Trypsin EDTA
    Description:
    Trypsin EDTA 0 04 0 03
    Catalog Number:
    c-41000
    Price:
    None
    Applications:
    Trypsin is still the most widely employed enzyme used for cell detachment. PromoCell´s Trypsin Solutions exhibit standardized enzyme activity and contain trypsin from extra-pure lots. This Trypsin/EDTA Solution has a ratio of 0.04% trypsin and 0.03% EDTA and is available in different sizes.
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    Millipore trypsin edta
    Trypsin EDTA
    Trypsin EDTA 0 04 0 03
    https://www.bioz.com/result/trypsin edta/product/Millipore
    Average 99 stars, based on 363 article reviews
    Price from $9.99 to $1999.99
    trypsin edta - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01146-08

    Inhibition of KSHV infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.
    Figure Legend Snippet: Inhibition of KSHV infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.

    Techniques Used: Inhibition, Infection, Incubation, Fluorescence, Microscopy, Standard Deviation, Binding Assay, Concentration Assay, Labeling, Purification, Isolation, Polymerase Chain Reaction

    ( A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 μg/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concentration of [ 3 H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [ 3 H]thymidine-labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radioactivity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation (SD) of three independent experiments. (B and C) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV DNA internalization. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h. Unbound virus was removed by washing the cells, and the cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus. The cells were washed, DNA was extracted, and the internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) Effect of CD98 ligand, galectin 3, on KSHV DNA internalization. HMVEC-d were first incubated with the CD98 ligand galectin at 5- and 10-μg/ml concentrations for 1 h at 4°C. The cells were washed and then infected with KSHV at an MOI of 10 for 2 h. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments. KSHV DNA internalization in the absence of any treatment was considered to be 100%.
    Figure Legend Snippet: ( A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 μg/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concentration of [ 3 H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [ 3 H]thymidine-labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radioactivity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation (SD) of three independent experiments. (B and C) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV DNA internalization. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h. Unbound virus was removed by washing the cells, and the cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus. The cells were washed, DNA was extracted, and the internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) Effect of CD98 ligand, galectin 3, on KSHV DNA internalization. HMVEC-d were first incubated with the CD98 ligand galectin at 5- and 10-μg/ml concentrations for 1 h at 4°C. The cells were washed and then infected with KSHV at an MOI of 10 for 2 h. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments. KSHV DNA internalization in the absence of any treatment was considered to be 100%.

    Techniques Used: Binding Assay, Incubation, Concentration Assay, Labeling, Radioactivity, Inhibition, Standard Deviation, Infection, Amplification, Polymerase Chain Reaction, Generated, Clone Assay

    2) Product Images from "Epithelial cells respond to proteolytic and non-proteolytic detachment by enhancing interleukin-6 responses"

    Article Title: Epithelial cells respond to proteolytic and non-proteolytic detachment by enhancing interleukin-6 responses

    Journal: Immunology

    doi: 10.1046/j.0019-2805.2001.01352.x

    Proteolytic detachment of IEC-6 cells enhances unstimulated and interleukin (IL)-1β-stimulated IL-6 secretion. IEC-6 cells (2·5 × 10 5 cells/well) were cultured for 2 days prior to washing and treatment with DMEM containing 5% fetal calf serum (FCS)±recombinant human (rh)IL-1β (untreated), 5% FCS DMEM with trypsin and EDTA±rhIL-1β (trypsin added), or trypsin and EDTA for 10 min, and then 5% FCS DMEM±rhIL-1β was added (to inhibit the trypsin and EDTA) and the detached cells were transferred to dBSA-coated wells (detached). After 24 hr, the supernatants were collected for IL-6 determination. (a) The effect on unstimulated cells and (b) the effect on rhIL-1β (1 ng/ml)-stimulated cells from the same experiment. Data represent the mean±SD values for triplicate wells from one experiment, representative of three separate experiments. *Significant difference from the untreated cells ( P ≤ 0·05).
    Figure Legend Snippet: Proteolytic detachment of IEC-6 cells enhances unstimulated and interleukin (IL)-1β-stimulated IL-6 secretion. IEC-6 cells (2·5 × 10 5 cells/well) were cultured for 2 days prior to washing and treatment with DMEM containing 5% fetal calf serum (FCS)±recombinant human (rh)IL-1β (untreated), 5% FCS DMEM with trypsin and EDTA±rhIL-1β (trypsin added), or trypsin and EDTA for 10 min, and then 5% FCS DMEM±rhIL-1β was added (to inhibit the trypsin and EDTA) and the detached cells were transferred to dBSA-coated wells (detached). After 24 hr, the supernatants were collected for IL-6 determination. (a) The effect on unstimulated cells and (b) the effect on rhIL-1β (1 ng/ml)-stimulated cells from the same experiment. Data represent the mean±SD values for triplicate wells from one experiment, representative of three separate experiments. *Significant difference from the untreated cells ( P ≤ 0·05).

    Techniques Used: Cell Culture, Recombinant

    3) Product Images from "Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages"

    Article Title: Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages

    Journal: Journal of Clinical Investigation

    doi:

    Chlamydial and human HSP 60s induce E-selectin production by endothelial cells. ( top ) ECs were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (5 μg/ml), human HSP 60 (5 μg/ml), E. coli LPS (1 μg/ml), or inactivated C. pneumoniae (10 7 U/ml), for 6 h. Cells were harvested by treatment with trypsin-EDTA and then stained with antibody against E-selectin ( solid histograms ) or with mouse IgGs (as isotype control; open histograms ). Chlamydial HSP 60 and human HSP 60 had a similar effect on E-selectin production as E. coli LPS. Inactivated C. pneumoniae did not elicit E-selectin expression by ECs. ( bottom ) Before incubation, reagents were heat-treated by boiling for 20 min. Heat treatment abolished the effect on E-selectin by chlamydial HSP 60 and human HSP 60, but did not modify the effect of thermostable E. coli LPS. Three independent experiments showed similar results. EC , endothelial cells; HSP , heat shock protein; LPS , lipopolysaccharide.
    Figure Legend Snippet: Chlamydial and human HSP 60s induce E-selectin production by endothelial cells. ( top ) ECs were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (5 μg/ml), human HSP 60 (5 μg/ml), E. coli LPS (1 μg/ml), or inactivated C. pneumoniae (10 7 U/ml), for 6 h. Cells were harvested by treatment with trypsin-EDTA and then stained with antibody against E-selectin ( solid histograms ) or with mouse IgGs (as isotype control; open histograms ). Chlamydial HSP 60 and human HSP 60 had a similar effect on E-selectin production as E. coli LPS. Inactivated C. pneumoniae did not elicit E-selectin expression by ECs. ( bottom ) Before incubation, reagents were heat-treated by boiling for 20 min. Heat treatment abolished the effect on E-selectin by chlamydial HSP 60 and human HSP 60, but did not modify the effect of thermostable E. coli LPS. Three independent experiments showed similar results. EC , endothelial cells; HSP , heat shock protein; LPS , lipopolysaccharide.

    Techniques Used: Incubation, Staining, Expressing

    4) Product Images from "Soluble matrix protein is a potent modulator of mesenchymal stem cell performance"

    Article Title: Soluble matrix protein is a potent modulator of mesenchymal stem cell performance

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1812951116

    Integrin-mediated effects of tropoelastin on MSC adhesion, spreading, and proliferation. ( A ) Cell adhesion to substrate-bound tropoelastin in the presence of EDTA. ( B ) Cell binding to tropoelastin in cation-free buffer with increasing doses of exogenous Mg 2+ , Ca 2+ , and Mn 2+ divalent cations. ( C – E ) Cell spreading on tropoelastin with increasing concentrations of an ( C ) anti-αvβ5, ( D ) anti-αvβ3, or ( E ) pan anti-αv integrin antibody. Cell spreading on fibronectin with and without the anti-αv integrin antibody is shown as a control. ( F ) Cell spreading on tropoelastin in the presence of optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, combined anti-αvβ3 and anti-αvβ5, and anti-αv integrin antibodies. Cell spreading on TCP and that on tropoelastin in the absence of antibodies or with a nonspecific mouse IgG antibody are also included as controls. Asterisks above the data columns refer to statistical differences from the no-antibody control. ( G ) Representative images of MSC spreading on tropoelastin, with and without integrin-blocking antibodies. (Scale bar: 100 μm.) ( H ) Confocal microscope images of MSCs adhered on tropoelastin- or BSA-coated TCP, stained for focal adhesion vinculin (green) and cell nuclei (blue). The relative density of focal adhesion staining per cell is indicated. (Scale bar: 20 μm.) ( I and J ) MSC proliferation in the presence of ( I ) FGFR and ( J ) integrin inhibitors. Cells were grown on TCP in normal media, in media with 20 μg/mL tropoelastin, or in bFGF-supplemented media for 7 d. ( I ) Increasing doses of the FGFR inhibitor, SU-5402, were added to the media during the proliferation period. Cell numbers were normalized against samples without SU-5402. Cell numbers in media containing tropoelastin or bFGF were compared with those in normal media at each inhibitor concentration to account for the nonspecific toxicity of SU-5402. ( J ) Optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, anti-αvβ5 and anti-αvβ3, or anti-αv were added to the media over 7 d. Controls without antibodies or with an antibody against a nonexpressed integrin (anti-β8) were included. Green arrows indicate cells grown in the presence of tropoelastin and αv integrin subunit antibodies. Asterisks above individual columns denote significant differences from cells in normal media at each antibody condition. ( K ) MSC proliferation after 7 d in the presence of an FAK inhibitor (FAK inhibitor 14) or a PKB/AKT inhibitor (perifosine). Cell numbers were normalized against uninhibited samples. Asterisks above individual columns represent comparison with the no-inhibitor control. * P
    Figure Legend Snippet: Integrin-mediated effects of tropoelastin on MSC adhesion, spreading, and proliferation. ( A ) Cell adhesion to substrate-bound tropoelastin in the presence of EDTA. ( B ) Cell binding to tropoelastin in cation-free buffer with increasing doses of exogenous Mg 2+ , Ca 2+ , and Mn 2+ divalent cations. ( C – E ) Cell spreading on tropoelastin with increasing concentrations of an ( C ) anti-αvβ5, ( D ) anti-αvβ3, or ( E ) pan anti-αv integrin antibody. Cell spreading on fibronectin with and without the anti-αv integrin antibody is shown as a control. ( F ) Cell spreading on tropoelastin in the presence of optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, combined anti-αvβ3 and anti-αvβ5, and anti-αv integrin antibodies. Cell spreading on TCP and that on tropoelastin in the absence of antibodies or with a nonspecific mouse IgG antibody are also included as controls. Asterisks above the data columns refer to statistical differences from the no-antibody control. ( G ) Representative images of MSC spreading on tropoelastin, with and without integrin-blocking antibodies. (Scale bar: 100 μm.) ( H ) Confocal microscope images of MSCs adhered on tropoelastin- or BSA-coated TCP, stained for focal adhesion vinculin (green) and cell nuclei (blue). The relative density of focal adhesion staining per cell is indicated. (Scale bar: 20 μm.) ( I and J ) MSC proliferation in the presence of ( I ) FGFR and ( J ) integrin inhibitors. Cells were grown on TCP in normal media, in media with 20 μg/mL tropoelastin, or in bFGF-supplemented media for 7 d. ( I ) Increasing doses of the FGFR inhibitor, SU-5402, were added to the media during the proliferation period. Cell numbers were normalized against samples without SU-5402. Cell numbers in media containing tropoelastin or bFGF were compared with those in normal media at each inhibitor concentration to account for the nonspecific toxicity of SU-5402. ( J ) Optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, anti-αvβ5 and anti-αvβ3, or anti-αv were added to the media over 7 d. Controls without antibodies or with an antibody against a nonexpressed integrin (anti-β8) were included. Green arrows indicate cells grown in the presence of tropoelastin and αv integrin subunit antibodies. Asterisks above individual columns denote significant differences from cells in normal media at each antibody condition. ( K ) MSC proliferation after 7 d in the presence of an FAK inhibitor (FAK inhibitor 14) or a PKB/AKT inhibitor (perifosine). Cell numbers were normalized against uninhibited samples. Asterisks above individual columns represent comparison with the no-inhibitor control. * P

    Techniques Used: Binding Assay, Blocking Assay, Microscopy, Staining, Concentration Assay

    5) Product Images from "Conditional knockout of N-Myc and STAT Interactor Disrupts Normal Mammary Development and Enhances Metastatic Ability of Mammary Tumors"

    Article Title: Conditional knockout of N-Myc and STAT Interactor Disrupts Normal Mammary Development and Enhances Metastatic Ability of Mammary Tumors

    Journal: Oncogene

    doi: 10.1038/s41388-017-0037-7

    Carcinogen and MMTV-Neu induced Nmi null tumors exhibit invasive morphological features. a Schematic for establishing 3D organoid cultures: tumors from control and knockout models were excised and digested using collagenase (10 mg/mL) and pronase (0.1 mg/mL) at 37 °C to separate tumor cells from the extracellular matrix. Organoids from tumors were overlaid onto 3D matrix (cultrex) in chamber slides (Millipore). b Morphologic behavior of control and Nmi knockout tumor cells was studied in 2 or 3-dimensional growth conditions. For 2D growth, tumor cells were allowed to attach to culture dishes and imaged prior to passaging. The 3D organoid cultures were established as described in the schematic a . Photomicrographs show phase contrast images of 2D and 3D cultures. The 3D culture images were captured after 14 days of culture to examine invasive processes. Scale bar = 100 μM for 2D cultures and 25 μM for 3D cultures. Green arrows in 2D images indicate smooth/cohesive edges of colonies whereas red arrows indicate non cohesive/serrated edges. Branches/organoid in 3D culture were counted and represented as bar graphs * p = 0.002; N = 6 ko mice and 4 cntrl mice (MMTV-Neu) and * p = 0.0001; N = 20 organoids per group (DMBA/MPA). c Organoid cultures from MMTV-Neu Nmi fl/fl and MMTV-Neu Nmi −/− were stained for fibrillar actin (phalloidin) to visualize invasive processes. DAPI was used to stain nuclei. Representative confocal images of organoids from control and knockout models are presented. d Tumor cells from MMTV-Neu control and knockout mice were separated into single cells using 0.25% trypsin/EDTA and dispase II (5 mg/ml) and placed (50,000 cells) into Boyden chambers using fibronectin (10 µg/ml) as a chemoattractant. Cells were allowed 24 h to invade through the matrigel coated chambers, fixed with 4% PFA and stained with crystal violet (the experiment was repeated independently) N = 5 control tumors and 4 knockout tumors. Quantification of invasion is a measure of the percentage of cells that invaded. * p = 0.001
    Figure Legend Snippet: Carcinogen and MMTV-Neu induced Nmi null tumors exhibit invasive morphological features. a Schematic for establishing 3D organoid cultures: tumors from control and knockout models were excised and digested using collagenase (10 mg/mL) and pronase (0.1 mg/mL) at 37 °C to separate tumor cells from the extracellular matrix. Organoids from tumors were overlaid onto 3D matrix (cultrex) in chamber slides (Millipore). b Morphologic behavior of control and Nmi knockout tumor cells was studied in 2 or 3-dimensional growth conditions. For 2D growth, tumor cells were allowed to attach to culture dishes and imaged prior to passaging. The 3D organoid cultures were established as described in the schematic a . Photomicrographs show phase contrast images of 2D and 3D cultures. The 3D culture images were captured after 14 days of culture to examine invasive processes. Scale bar = 100 μM for 2D cultures and 25 μM for 3D cultures. Green arrows in 2D images indicate smooth/cohesive edges of colonies whereas red arrows indicate non cohesive/serrated edges. Branches/organoid in 3D culture were counted and represented as bar graphs * p = 0.002; N = 6 ko mice and 4 cntrl mice (MMTV-Neu) and * p = 0.0001; N = 20 organoids per group (DMBA/MPA). c Organoid cultures from MMTV-Neu Nmi fl/fl and MMTV-Neu Nmi −/− were stained for fibrillar actin (phalloidin) to visualize invasive processes. DAPI was used to stain nuclei. Representative confocal images of organoids from control and knockout models are presented. d Tumor cells from MMTV-Neu control and knockout mice were separated into single cells using 0.25% trypsin/EDTA and dispase II (5 mg/ml) and placed (50,000 cells) into Boyden chambers using fibronectin (10 µg/ml) as a chemoattractant. Cells were allowed 24 h to invade through the matrigel coated chambers, fixed with 4% PFA and stained with crystal violet (the experiment was repeated independently) N = 5 control tumors and 4 knockout tumors. Quantification of invasion is a measure of the percentage of cells that invaded. * p = 0.001

    Techniques Used: Knock-Out, Passaging, Mouse Assay, Staining

    6) Product Images from "Conditional knockout of N-Myc and STAT Interactor Disrupts Normal Mammary Development and Enhances Metastatic Ability of Mammary Tumors"

    Article Title: Conditional knockout of N-Myc and STAT Interactor Disrupts Normal Mammary Development and Enhances Metastatic Ability of Mammary Tumors

    Journal: Oncogene

    doi: 10.1038/s41388-017-0037-7

    Carcinogen and MMTV-Neu induced Nmi null tumors exhibit invasive morphological features. a Schematic for establishing 3D organoid cultures: tumors from control and knockout models were excised and digested using collagenase (10 mg/mL) and pronase (0.1 mg/mL) at 37 °C to separate tumor cells from the extracellular matrix. Organoids from tumors were overlaid onto 3D matrix (cultrex) in chamber slides (Millipore). b Morphologic behavior of control and Nmi knockout tumor cells was studied in 2 or 3-dimensional growth conditions. For 2D growth, tumor cells were allowed to attach to culture dishes and imaged prior to passaging. The 3D organoid cultures were established as described in the schematic a . Photomicrographs show phase contrast images of 2D and 3D cultures. The 3D culture images were captured after 14 days of culture to examine invasive processes. Scale bar = 100 μM for 2D cultures and 25 μM for 3D cultures. Green arrows in 2D images indicate smooth/cohesive edges of colonies whereas red arrows indicate non cohesive/serrated edges. Branches/organoid in 3D culture were counted and represented as bar graphs * p = 0.002; N = 6 ko mice and 4 cntrl mice (MMTV-Neu) and * p = 0.0001; N = 20 organoids per group (DMBA/MPA). c Organoid cultures from MMTV-Neu Nmi fl/fl and MMTV-Neu Nmi −/− were stained for fibrillar actin (phalloidin) to visualize invasive processes. DAPI was used to stain nuclei. Representative confocal images of organoids from control and knockout models are presented. d Tumor cells from MMTV-Neu control and knockout mice were separated into single cells using 0.25% trypsin/EDTA and dispase II (5 mg/ml) and placed (50,000 cells) into Boyden chambers using fibronectin (10 µg/ml) as a chemoattractant. Cells were allowed 24 h to invade through the matrigel coated chambers, fixed with 4% PFA and stained with crystal violet (the experiment was repeated independently) N = 5 control tumors and 4 knockout tumors. Quantification of invasion is a measure of the percentage of cells that invaded. * p = 0.001
    Figure Legend Snippet: Carcinogen and MMTV-Neu induced Nmi null tumors exhibit invasive morphological features. a Schematic for establishing 3D organoid cultures: tumors from control and knockout models were excised and digested using collagenase (10 mg/mL) and pronase (0.1 mg/mL) at 37 °C to separate tumor cells from the extracellular matrix. Organoids from tumors were overlaid onto 3D matrix (cultrex) in chamber slides (Millipore). b Morphologic behavior of control and Nmi knockout tumor cells was studied in 2 or 3-dimensional growth conditions. For 2D growth, tumor cells were allowed to attach to culture dishes and imaged prior to passaging. The 3D organoid cultures were established as described in the schematic a . Photomicrographs show phase contrast images of 2D and 3D cultures. The 3D culture images were captured after 14 days of culture to examine invasive processes. Scale bar = 100 μM for 2D cultures and 25 μM for 3D cultures. Green arrows in 2D images indicate smooth/cohesive edges of colonies whereas red arrows indicate non cohesive/serrated edges. Branches/organoid in 3D culture were counted and represented as bar graphs * p = 0.002; N = 6 ko mice and 4 cntrl mice (MMTV-Neu) and * p = 0.0001; N = 20 organoids per group (DMBA/MPA). c Organoid cultures from MMTV-Neu Nmi fl/fl and MMTV-Neu Nmi −/− were stained for fibrillar actin (phalloidin) to visualize invasive processes. DAPI was used to stain nuclei. Representative confocal images of organoids from control and knockout models are presented. d Tumor cells from MMTV-Neu control and knockout mice were separated into single cells using 0.25% trypsin/EDTA and dispase II (5 mg/ml) and placed (50,000 cells) into Boyden chambers using fibronectin (10 µg/ml) as a chemoattractant. Cells were allowed 24 h to invade through the matrigel coated chambers, fixed with 4% PFA and stained with crystal violet (the experiment was repeated independently) N = 5 control tumors and 4 knockout tumors. Quantification of invasion is a measure of the percentage of cells that invaded. * p = 0.001

    Techniques Used: Knock-Out, Passaging, Mouse Assay, Staining

    7) Product Images from "Acanthamoeba castellanii Promotion of In Vitro Survival and Transmission of Coxsackie B3 Viruses"

    Article Title: Acanthamoeba castellanii Promotion of In Vitro Survival and Transmission of Coxsackie B3 Viruses

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.5.4.665-671.2006

    Virus inactivation in A. castellanii trophozoites infected with CVB-3 (A) or ECHO-31 viruses (B) after exposure to 0.25% trypsin-EDTA for 10 min at 37°C. Values are means ± standard errors from at least six experiments. *, P
    Figure Legend Snippet: Virus inactivation in A. castellanii trophozoites infected with CVB-3 (A) or ECHO-31 viruses (B) after exposure to 0.25% trypsin-EDTA for 10 min at 37°C. Values are means ± standard errors from at least six experiments. *, P

    Techniques Used: Infection

    8) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.2.1191-1206.2005

    HHV-8-induced MT dynamics influence the nuclear delivery of HHV-8 DNA. (A) Purification of infected cell nuclei. Uninfected and HHV-8-infected HFF cells were washed with PBS, treated with trypsin-EDTA to remove noninternalized virus, washed, suspended
    Figure Legend Snippet: HHV-8-induced MT dynamics influence the nuclear delivery of HHV-8 DNA. (A) Purification of infected cell nuclei. Uninfected and HHV-8-infected HFF cells were washed with PBS, treated with trypsin-EDTA to remove noninternalized virus, washed, suspended

    Techniques Used: Purification, Infection

    9) Product Images from "Promoting Long-Term Survival of Insulin-Producing Cell Grafts That Differentiate from Adipose Tissue-Derived Stem Cells to Cure Type 1 Diabetes"

    Article Title: Promoting Long-Term Survival of Insulin-Producing Cell Grafts That Differentiate from Adipose Tissue-Derived Stem Cells to Cure Type 1 Diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029706

    Impaired IPCC, but not islet, graft survival in syngeneic recipient mice. Islets freshly isolated from B6 donors or IPCCs that differentiated from B6-derived ASCs were transplanted under kidney capsule of either B6.Rag-/- or WT B6 recipient mice that were rendered diabetic by a single injection of streptozotocin before transplantation. Islet and IPCC syngraft rejection was observed ( A ). In separate groups, transplanted islets or IPCCs were retrieved and dissociated with trypsin-EDTA one week after transplantation. Cells were then stained with PE-conjugated PDX-1 and their apoptosis was measured by a TUNEL method ( B ). Histograms are gated on PDX-1-positive β cell population. One representative of three separate experiments is shown.
    Figure Legend Snippet: Impaired IPCC, but not islet, graft survival in syngeneic recipient mice. Islets freshly isolated from B6 donors or IPCCs that differentiated from B6-derived ASCs were transplanted under kidney capsule of either B6.Rag-/- or WT B6 recipient mice that were rendered diabetic by a single injection of streptozotocin before transplantation. Islet and IPCC syngraft rejection was observed ( A ). In separate groups, transplanted islets or IPCCs were retrieved and dissociated with trypsin-EDTA one week after transplantation. Cells were then stained with PE-conjugated PDX-1 and their apoptosis was measured by a TUNEL method ( B ). Histograms are gated on PDX-1-positive β cell population. One representative of three separate experiments is shown.

    Techniques Used: Mouse Assay, Isolation, Derivative Assay, Injection, Transplantation Assay, Staining, TUNEL Assay

    10) Product Images from "DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions"

    Article Title: DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20032206

    DNAM-1 recognizes junctional ligands expressed on endothelial cells. (a) HUVECs were analyzed by one-color immunofluorescence and FACS ® analysis with Nec2-Fc, PVR-Fc, DNAM-1–Fc, and N4vtr-Fc (negative control) followed by FITC-conjugated goat anti–human second reagents. Gray profiles indicate cells incubated with the second reagent only. All soluble proteins were used at 20 μg/ml. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (b) Immunofluorescence microscopy analysis of HUVECs using 20 μg/ml DNAM-1–Fc followed by FITC-conjugated goat anti–human second reagents. No staining was revealed with Nec2-Fc. The DNAM-1–Fc staining delineates the junctional systems between adjacent cells suggesting that DNAM-1 interacts with ligands localized at endothelial cell junctions. Similar results were obtained on live confluent HUVECs (not depicted).
    Figure Legend Snippet: DNAM-1 recognizes junctional ligands expressed on endothelial cells. (a) HUVECs were analyzed by one-color immunofluorescence and FACS ® analysis with Nec2-Fc, PVR-Fc, DNAM-1–Fc, and N4vtr-Fc (negative control) followed by FITC-conjugated goat anti–human second reagents. Gray profiles indicate cells incubated with the second reagent only. All soluble proteins were used at 20 μg/ml. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (b) Immunofluorescence microscopy analysis of HUVECs using 20 μg/ml DNAM-1–Fc followed by FITC-conjugated goat anti–human second reagents. No staining was revealed with Nec2-Fc. The DNAM-1–Fc staining delineates the junctional systems between adjacent cells suggesting that DNAM-1 interacts with ligands localized at endothelial cell junctions. Similar results were obtained on live confluent HUVECs (not depicted).

    Techniques Used: Immunofluorescence, FACS, Negative Control, Incubation, Microscopy, Staining

    Analysis of the cell surface expression of DNAM-1, PVR, and Nectin-2 on monocytes and primary vascular endothelial cells. (a) Fresh PBMCs were gated on monocytes on the basis of both size and granularity. Cells were analyzed by two-color immunofluorescence and FACS ® analysis with anti-CD14 mAb in combination with anti–DNAM-1 (FS.123), anti-PVR (PV.404), or anti–Nectin-2 (R2.477) mAbs followed by FITC- or PE-conjugated goat anti–mouse second reagents. (b) HUVECs were analyzed by one-color immunofluorescence and FACS ® analysis with anti–DNAM-1 (FS123), anti-PVR (PV.404), anti–Nectin-2 (R2.477), or anti–PECAM-1 (JC/70A) mAbs followed by FITC-conjugated goat anti–mouse second reagents. Gray profiles indicate cells incubated with the second reagent only. We previously controlled that trypsin treatment does not affect cell surface expression of Nectins. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (c) Localization of Nectin-2 and PVR on primary endothelial cells. HUVECs were fixed; incubated with 2 μg/ml of anti–Nectin-2 (R2.477), anti-PVR (PV.404), and anti–PECAM-1 (JC/70), followed by Alexa-488–labeled goat anti–mouse second reagent; and analyzed by immunofluorescence microscopy. Staining was similar on live cells (not depicted). (d) HUVECs were fixed, permeabilized, and stained with the anti–Nectin-2 (R2.477) mAb and the anti–β-catenin rabbit antiserum followed by Alexa-488–labeled goat anti–mouse antibody and TRITC-labeled goat anti–rabbit second reagents, respectively. Cells were analyzed by immunofluorescence microscopy.
    Figure Legend Snippet: Analysis of the cell surface expression of DNAM-1, PVR, and Nectin-2 on monocytes and primary vascular endothelial cells. (a) Fresh PBMCs were gated on monocytes on the basis of both size and granularity. Cells were analyzed by two-color immunofluorescence and FACS ® analysis with anti-CD14 mAb in combination with anti–DNAM-1 (FS.123), anti-PVR (PV.404), or anti–Nectin-2 (R2.477) mAbs followed by FITC- or PE-conjugated goat anti–mouse second reagents. (b) HUVECs were analyzed by one-color immunofluorescence and FACS ® analysis with anti–DNAM-1 (FS123), anti-PVR (PV.404), anti–Nectin-2 (R2.477), or anti–PECAM-1 (JC/70A) mAbs followed by FITC-conjugated goat anti–mouse second reagents. Gray profiles indicate cells incubated with the second reagent only. We previously controlled that trypsin treatment does not affect cell surface expression of Nectins. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (c) Localization of Nectin-2 and PVR on primary endothelial cells. HUVECs were fixed; incubated with 2 μg/ml of anti–Nectin-2 (R2.477), anti-PVR (PV.404), and anti–PECAM-1 (JC/70), followed by Alexa-488–labeled goat anti–mouse second reagent; and analyzed by immunofluorescence microscopy. Staining was similar on live cells (not depicted). (d) HUVECs were fixed, permeabilized, and stained with the anti–Nectin-2 (R2.477) mAb and the anti–β-catenin rabbit antiserum followed by Alexa-488–labeled goat anti–mouse antibody and TRITC-labeled goat anti–rabbit second reagents, respectively. Cells were analyzed by immunofluorescence microscopy.

    Techniques Used: Expressing, Immunofluorescence, FACS, Incubation, Labeling, Microscopy, Staining

    11) Product Images from "A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier"

    Article Title: A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier

    Journal: mSphere

    doi: 10.1128/mSphere.00206-17

    Apical junctions restrict RNA virus infection of 3-D-cultured HBMEC. (A) RT-qPCR for analysis of VSV, DENV, ZIKV C , or ZIKV B infection viral RNA (vRNA) in 2-D, 3-D, or 3-D tryp cultures of HBMEC. (B) RT-qPCR for DENV or ZIKV C vRNA in 2-D, 3-D, or 3-D tryp cultures of HBMEC treated with 10 mM EDTA for 1 h prior to infection. (C) RT-qPCR for ZIKV vRNA in 2-D, 3-D, or 3-D tryp cultures of HBMEC that were treated with 100 ng/ml TNF-α for 24 h prior to infection with ZIKV B for an additional 48 h (in the presence of TNF-α). (D) ZIKV infectious titers from mock- or TNF-α-treated 3-D-cultured HBMEC. (E) Confocal micrographs of mock- or TNF-α-treated 3-D cultures of HBMEC stained for actin (green). DAPI-stained nuclei are shown in blue. (F) Schematic for monocyte transmigration assay using ZIKV B -infected monocytes (shown in green), 2-D or 3-D tryp HBMEC plated in the apical chamber, and primary human astrocytes plated in the basolateral compartment. (G) Quantification (performed in triplicate) of the percentages of ZIKV B -infected or control uninfected monocytes transmigrating from the apical to basolateral chambers across 2-D or two independent 3-D tryp preparations. (H) Confocal micrographs of CFDA-labeled ZIKV B -infected THP-1 cells (green) 24 h following their addition to the apical chambers of Transwell inserts containing 2-D or 3-D tryp cultures stained for actin (in red). DAPI-stained nuclei are shown in blue. THP-1 cells that remained in the apical surfaces of 2-D cells are shown with gray arrows, and THP-1 cells in the 3-D tryp panels that were in the process of transmigrating the endothelial monolayer are shown with white arrows. Images are representative of cells isolated from two independent 3-D tryp preparations in experiments performed in triplicate. Data in panels A to D and G are shown as means ± standard deviations and are normalized as a percentage of 2-D results (A), mock-treated cells (B and C), or percent input cells (G) (*, P
    Figure Legend Snippet: Apical junctions restrict RNA virus infection of 3-D-cultured HBMEC. (A) RT-qPCR for analysis of VSV, DENV, ZIKV C , or ZIKV B infection viral RNA (vRNA) in 2-D, 3-D, or 3-D tryp cultures of HBMEC. (B) RT-qPCR for DENV or ZIKV C vRNA in 2-D, 3-D, or 3-D tryp cultures of HBMEC treated with 10 mM EDTA for 1 h prior to infection. (C) RT-qPCR for ZIKV vRNA in 2-D, 3-D, or 3-D tryp cultures of HBMEC that were treated with 100 ng/ml TNF-α for 24 h prior to infection with ZIKV B for an additional 48 h (in the presence of TNF-α). (D) ZIKV infectious titers from mock- or TNF-α-treated 3-D-cultured HBMEC. (E) Confocal micrographs of mock- or TNF-α-treated 3-D cultures of HBMEC stained for actin (green). DAPI-stained nuclei are shown in blue. (F) Schematic for monocyte transmigration assay using ZIKV B -infected monocytes (shown in green), 2-D or 3-D tryp HBMEC plated in the apical chamber, and primary human astrocytes plated in the basolateral compartment. (G) Quantification (performed in triplicate) of the percentages of ZIKV B -infected or control uninfected monocytes transmigrating from the apical to basolateral chambers across 2-D or two independent 3-D tryp preparations. (H) Confocal micrographs of CFDA-labeled ZIKV B -infected THP-1 cells (green) 24 h following their addition to the apical chambers of Transwell inserts containing 2-D or 3-D tryp cultures stained for actin (in red). DAPI-stained nuclei are shown in blue. THP-1 cells that remained in the apical surfaces of 2-D cells are shown with gray arrows, and THP-1 cells in the 3-D tryp panels that were in the process of transmigrating the endothelial monolayer are shown with white arrows. Images are representative of cells isolated from two independent 3-D tryp preparations in experiments performed in triplicate. Data in panels A to D and G are shown as means ± standard deviations and are normalized as a percentage of 2-D results (A), mock-treated cells (B and C), or percent input cells (G) (*, P

    Techniques Used: Infection, Cell Culture, Quantitative RT-PCR, Staining, Transmigration Assay, Labeling, Isolation

    Related Articles

    Centrifugation:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

    Article Title: Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium
    Article Snippet: .. Confluent cells were retrypsinized (5-7 minutes at 37 °C) using a trypsin-EDTA buffer (0.1%, Sigma-Aldrich, St. Louis, MO), followed by centrifugation (125 × g, 7 min). ..

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus
    Article Snippet: .. Briefly, cells infected with HHV-8 were collected at different times postinfection (p.i.), washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures. .. The nuclei from one 25-cm2 flask were resuspended in nucleus homogenization buffer (250 mM sucrose, 5 mM MgCl2 · 6H2 O, 25 mM KCl, 20 mM Tricine-KOH, 7.8), and 60% iodixanol (Optiprep; Axis-Shield, Oslo, Norway) was added to a final concentration of 25% iodixanol.

    Isolation:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

    Knock-Out:

    Article Title: Conditional knockout of N-Myc and STAT Interactor Disrupts Normal Mammary Development and Enhances Metastatic Ability of Mammary Tumors
    Article Snippet: .. Epithelial cells from mammary tumors were harvested from MMTV-Neu Nmi knockout and control mice and dissociated into single cells using 0.25% trypsin/EDTA and dispase II (5 mg/ml, Sigma). ..

    Mouse Assay:

    Article Title: Conditional knockout of N-Myc and STAT Interactor Disrupts Normal Mammary Development and Enhances Metastatic Ability of Mammary Tumors
    Article Snippet: .. Epithelial cells from mammary tumors were harvested from MMTV-Neu Nmi knockout and control mice and dissociated into single cells using 0.25% trypsin/EDTA and dispase II (5 mg/ml, Sigma). ..

    Incubation:

    Article Title: A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier
    Article Snippet: .. HBMEC propagated as described above were harvested in 0.05% trypsin–EDTA and incubated with ~300 mg collagen-coated Cytodex-3 beads (Sigma) at 6 × 106 cells/300 mg beads. .. After a brief (~30-to-60-min) static incubation at 37°C, the bead/cell slurry was added to autoclavable 55-ml slow-turning lateral vessels (STLVs) and was attached to a rotating base (Synthecon) at 19 to 21 rpm to maintain the cells in suspension for the duration of the culture period (21 days).

    Infection:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus
    Article Snippet: .. Briefly, cells infected with HHV-8 were collected at different times postinfection (p.i.), washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures. .. The nuclei from one 25-cm2 flask were resuspended in nucleus homogenization buffer (250 mM sucrose, 5 mM MgCl2 · 6H2 O, 25 mM KCl, 20 mM Tricine-KOH, 7.8), and 60% iodixanol (Optiprep; Axis-Shield, Oslo, Norway) was added to a final concentration of 25% iodixanol.

    Staining:

    Article Title: Conditional knockout of N-Myc and STAT Interactor Disrupts Normal Mammary Development and Enhances Metastatic Ability of Mammary Tumors
    Article Snippet: .. Mammary epithelial cells were dissociated into single cells using 0.25% trypsin/EDTA and Dispase II (5 mg/ml, Sigma) and stained with with the following antibodies: CD24-PE, CD49f-FITC (BD Biosciences) CD61-APC, CD14-FITC, CD29- Alexa700, Sca-1-BV510, and CD117-BV405 (Biolegend, San Diego, CA, USA). ..

    Lysis:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus
    Article Snippet: .. Briefly, cells infected with HHV-8 were collected at different times postinfection (p.i.), washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures. .. The nuclei from one 25-cm2 flask were resuspended in nucleus homogenization buffer (250 mM sucrose, 5 mM MgCl2 · 6H2 O, 25 mM KCl, 20 mM Tricine-KOH, 7.8), and 60% iodixanol (Optiprep; Axis-Shield, Oslo, Norway) was added to a final concentration of 25% iodixanol.

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