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  • 99
    Millipore pronase
    Pronase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pronase/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pronase - by Bioz Stars, 2021-06
    99/100 stars
      Buy from Supplier

    86
    Roche pronase
    Vector uptake into a protease-resistant compartment and subsequent transfer. (A) GFP marking in (CD45 negative) 293T 2° cells following coculture with vector exposed SupT1 cells washed in media (shaded) or <t>pronase</t> wash (non-shaded). Error bars represent standard deviation between samples. (B) K562 cells were vector-exposed, pronase-washed and co-cultured with 293T cells. CD45-APC staining captured 90% of myeloid population, implying that %GFP positive 293T cells (C) may reflect a minor percentage of admixed residual K562 cells. (D) L1210 cells were exposed to vector at low MOI for 3 hours, followed by pronase wash, independent propagation for designated time ( x- axis), and coculture with 293T cells. Vector genomes in 293T cells were detected by FACS with exclusion of 1° cells by CD45 staining. (E) L1210 cells were exposed to vector for increasing lengths of time ( x -axis) and washed with pronase. RNA was collected, reverse transcribed with random hexamer primers, and quantitative real-time PCR was performed using primers to detect proviral GFP sequence (DNA) or dLTR sequence (vector genome RNA). (F) Densiometric analysis and p24 immunoblot (inset). Cell lysates prepared from vector-exposed, pronase-washed Jurkat cells, resolved on a 10% PAGE gel, probed with antibody against p24 or β-tubulin. (G) GFP-vpr labeled genomes (green), anti-p24 was fluorescently labeled AlexaFluor 647 (far-red), and colocalized GFP-vpr with p24 particles (yellow) were counted in each cell. The y-axis represents the percent of total cells counted with a given number of colocalized particles. (H, I) Representative images of Jurkat cells on day 1 and 4, respectively, after vector exposure.
    Pronase, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pronase/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pronase - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    86
    Merck KGaA pronase e
    Cultures of hepatic stellate cells isolated via iohexol density gradient centrifugation without or with subsequent cell sorting. Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on <t>pronase</t> and collagenase, followed by density gradient centrifugation in 8% iohexol. Cells were cultured directly after the gradient for one day (a) and four days (b), where Kupffer cells (indicated by arrows) can be found in the HSC culture. Highly pure HSC after additional fluorescence-activated cell sorting (FACS) after one (c) and four days of culture are shown (d). Expression analysis of desmin (e) and phalloidin (f) of HSC after four days of culture, indicating proper maturation of HSC.
    Pronase E, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pronase e/product/Merck KGaA
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pronase e - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    N/A
    Pronase rabbit polyclonal antibody Azide Free
      Buy from Supplier

    N/A
    IgG fraction of polyclonal rabbit antiserum to pronase from Streptomyces griseus This product is intended for use in precipitating and non precipitating antibody binding assays such as e g ELISA
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    N/A
    Affinity purified antibodies of polyclonal rabbit antiserum to pronase from Streptomyces griseus This product is intended for use in precipitating and non precipitating antibody binding assays such as e g
      Buy from Supplier

    N/A
    Biotin conjugated IgG fraction of polyclonal rabbit antiserum to pronase from steptomyces griseus This product is intended for use in precipitating and non precipitating antibody binding assays such as e
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    Image Search Results


    Vector uptake into a protease-resistant compartment and subsequent transfer. (A) GFP marking in (CD45 negative) 293T 2° cells following coculture with vector exposed SupT1 cells washed in media (shaded) or pronase wash (non-shaded). Error bars represent standard deviation between samples. (B) K562 cells were vector-exposed, pronase-washed and co-cultured with 293T cells. CD45-APC staining captured 90% of myeloid population, implying that %GFP positive 293T cells (C) may reflect a minor percentage of admixed residual K562 cells. (D) L1210 cells were exposed to vector at low MOI for 3 hours, followed by pronase wash, independent propagation for designated time ( x- axis), and coculture with 293T cells. Vector genomes in 293T cells were detected by FACS with exclusion of 1° cells by CD45 staining. (E) L1210 cells were exposed to vector for increasing lengths of time ( x -axis) and washed with pronase. RNA was collected, reverse transcribed with random hexamer primers, and quantitative real-time PCR was performed using primers to detect proviral GFP sequence (DNA) or dLTR sequence (vector genome RNA). (F) Densiometric analysis and p24 immunoblot (inset). Cell lysates prepared from vector-exposed, pronase-washed Jurkat cells, resolved on a 10% PAGE gel, probed with antibody against p24 or β-tubulin. (G) GFP-vpr labeled genomes (green), anti-p24 was fluorescently labeled AlexaFluor 647 (far-red), and colocalized GFP-vpr with p24 particles (yellow) were counted in each cell. The y-axis represents the percent of total cells counted with a given number of colocalized particles. (H, I) Representative images of Jurkat cells on day 1 and 4, respectively, after vector exposure.

    Journal: PLoS ONE

    Article Title: Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells

    doi: 10.1371/journal.pone.0006219

    Figure Lengend Snippet: Vector uptake into a protease-resistant compartment and subsequent transfer. (A) GFP marking in (CD45 negative) 293T 2° cells following coculture with vector exposed SupT1 cells washed in media (shaded) or pronase wash (non-shaded). Error bars represent standard deviation between samples. (B) K562 cells were vector-exposed, pronase-washed and co-cultured with 293T cells. CD45-APC staining captured 90% of myeloid population, implying that %GFP positive 293T cells (C) may reflect a minor percentage of admixed residual K562 cells. (D) L1210 cells were exposed to vector at low MOI for 3 hours, followed by pronase wash, independent propagation for designated time ( x- axis), and coculture with 293T cells. Vector genomes in 293T cells were detected by FACS with exclusion of 1° cells by CD45 staining. (E) L1210 cells were exposed to vector for increasing lengths of time ( x -axis) and washed with pronase. RNA was collected, reverse transcribed with random hexamer primers, and quantitative real-time PCR was performed using primers to detect proviral GFP sequence (DNA) or dLTR sequence (vector genome RNA). (F) Densiometric analysis and p24 immunoblot (inset). Cell lysates prepared from vector-exposed, pronase-washed Jurkat cells, resolved on a 10% PAGE gel, probed with antibody against p24 or β-tubulin. (G) GFP-vpr labeled genomes (green), anti-p24 was fluorescently labeled AlexaFluor 647 (far-red), and colocalized GFP-vpr with p24 particles (yellow) were counted in each cell. The y-axis represents the percent of total cells counted with a given number of colocalized particles. (H, I) Representative images of Jurkat cells on day 1 and 4, respectively, after vector exposure.

    Article Snippet: For pronase washes, cells were pelleted immediately following transduction and resuspended in 1 mg/ml pronase (Roche) for 10 minutes at 37°C followed by two washes in media containing 10% FBS.

    Techniques: Plasmid Preparation, Standard Deviation, Cell Culture, Staining, FACS, Random Hexamer Labeling, Real-time Polymerase Chain Reaction, Sequencing, Polyacrylamide Gel Electrophoresis, Labeling

    Inhibiting MVB formation abrogates 2° transfer. (A) Enumeration of GFP-vpr vector genomes (green) associated with select MVB markers (N-Rh-PE, red and CD63 tetraspanin, magenta) following exposure to 100 µM LY-294002. Cell aliquots were collected at 1 and 3 hours following exposure. (B) Top panels are representative images from untreated cells, bottom panels representative of treated cells. (C) Functional effect of LY-294002 on 2° transfer of vector genomes. Jurkat carrier cells were pretreated with escalating doses of LY-294002 as in (A,B) followed by vector exposure for 3 hours in the presence of the inhibitor, pronase wash, and 24 hour co-culture with 293T cells. Primary marking (gray), 2° transfer (black), and % efficiency of 2° transfer (red) are shown.

    Journal: PLoS ONE

    Article Title: Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells

    doi: 10.1371/journal.pone.0006219

    Figure Lengend Snippet: Inhibiting MVB formation abrogates 2° transfer. (A) Enumeration of GFP-vpr vector genomes (green) associated with select MVB markers (N-Rh-PE, red and CD63 tetraspanin, magenta) following exposure to 100 µM LY-294002. Cell aliquots were collected at 1 and 3 hours following exposure. (B) Top panels are representative images from untreated cells, bottom panels representative of treated cells. (C) Functional effect of LY-294002 on 2° transfer of vector genomes. Jurkat carrier cells were pretreated with escalating doses of LY-294002 as in (A,B) followed by vector exposure for 3 hours in the presence of the inhibitor, pronase wash, and 24 hour co-culture with 293T cells. Primary marking (gray), 2° transfer (black), and % efficiency of 2° transfer (red) are shown.

    Article Snippet: For pronase washes, cells were pelleted immediately following transduction and resuspended in 1 mg/ml pronase (Roche) for 10 minutes at 37°C followed by two washes in media containing 10% FBS.

    Techniques: Plasmid Preparation, Functional Assay, Co-Culture Assay

    Vector particles persist in an unprocessed state. (A) 293T 1° cells were incubated overnight in AZT, followed by 3-hour vector exposure in the presence of AZT, pronase wash, and co-culture (without AZT) with Jurkat 2° cells. GFP expression was determined in the 2° CD45+ (Jurkat) population by FACS. (B) Jurkat cells were exposed to vector, pronase washed, and placed in co-culture with 293T cells with (+/−) anti-VSV-G neutralizing antibody. GFP was examined by FACS. (C) VSV-G immunoblot of cell lysates prepared from from vector-exposed, pronase-washed Jurkat cells probed with antibody against VSV-G or β-tubulin. The positive control is neat vector; each lane corresponds to the day of (or after) vector exposure. (inset) Densiometric analysis of immunoblot (D) Jurkat cells were exposed to GFP-vpr tagged vector, washed with pronase, and propagated in culture for 4 additional days. The vector genomes are GFP-vpr labeled (green), and anti-VSV-G is fluorescently labeled with the 2° antibody AlexaFluor 647 (far-red). Colocalized GFP-vpr and p24 particles (yellow) were counted in each cell. The left y- axis represents the percent of total cells counted with a given number of colocalized particles. The right y- axis represents the percentage of particles associated with VSV-G envelope. (E, F) Representative images of Jurkat cell on day 1 and 4, respectively, after vector exposure. GFP-vpr particles (green), anti-VSV-G antiserum (magenta), and colocalized particles (white) are shown.

    Journal: PLoS ONE

    Article Title: Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells

    doi: 10.1371/journal.pone.0006219

    Figure Lengend Snippet: Vector particles persist in an unprocessed state. (A) 293T 1° cells were incubated overnight in AZT, followed by 3-hour vector exposure in the presence of AZT, pronase wash, and co-culture (without AZT) with Jurkat 2° cells. GFP expression was determined in the 2° CD45+ (Jurkat) population by FACS. (B) Jurkat cells were exposed to vector, pronase washed, and placed in co-culture with 293T cells with (+/−) anti-VSV-G neutralizing antibody. GFP was examined by FACS. (C) VSV-G immunoblot of cell lysates prepared from from vector-exposed, pronase-washed Jurkat cells probed with antibody against VSV-G or β-tubulin. The positive control is neat vector; each lane corresponds to the day of (or after) vector exposure. (inset) Densiometric analysis of immunoblot (D) Jurkat cells were exposed to GFP-vpr tagged vector, washed with pronase, and propagated in culture for 4 additional days. The vector genomes are GFP-vpr labeled (green), and anti-VSV-G is fluorescently labeled with the 2° antibody AlexaFluor 647 (far-red). Colocalized GFP-vpr and p24 particles (yellow) were counted in each cell. The left y- axis represents the percent of total cells counted with a given number of colocalized particles. The right y- axis represents the percentage of particles associated with VSV-G envelope. (E, F) Representative images of Jurkat cell on day 1 and 4, respectively, after vector exposure. GFP-vpr particles (green), anti-VSV-G antiserum (magenta), and colocalized particles (white) are shown.

    Article Snippet: For pronase washes, cells were pelleted immediately following transduction and resuspended in 1 mg/ml pronase (Roche) for 10 minutes at 37°C followed by two washes in media containing 10% FBS.

    Techniques: Plasmid Preparation, Incubation, Co-Culture Assay, Expressing, FACS, Positive Control, Labeling

    Vector particles associate with MVB markers. (A,B) Vector genomes associate with select MVB markers. Jurkat cells exposed to GFP-vpr vector (green) overnight, followed by pronase wash, and stain with antibodies against CD81, CD63, or N-Rh-PE (red), MHCII (magenta). Particles found associated with CD81 and MHCII, or N-Rh-PE and MHCII are white. (C) Live cell imaging of vector-exposed, pronase-washed 1° SupT1 cells (labeled with anti-CD81, Alexa Fluor 647, magenta) in co-culture with 2° 293T DsRed actin (red) expressing cells. Right hand panels lack the DsRed layer for improved visual clarity of otherwise identical frames. Genomes co-localized with tetraspanin are white (arrows, boxes). Genomes co-localized with DsRed actin are yellow. Deconvolution microscopy was performed on live cells by collecting series of z -stacks (0.5 µm) every minute for 10 minutes, elapsed time is indicated in black.

    Journal: PLoS ONE

    Article Title: Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells

    doi: 10.1371/journal.pone.0006219

    Figure Lengend Snippet: Vector particles associate with MVB markers. (A,B) Vector genomes associate with select MVB markers. Jurkat cells exposed to GFP-vpr vector (green) overnight, followed by pronase wash, and stain with antibodies against CD81, CD63, or N-Rh-PE (red), MHCII (magenta). Particles found associated with CD81 and MHCII, or N-Rh-PE and MHCII are white. (C) Live cell imaging of vector-exposed, pronase-washed 1° SupT1 cells (labeled with anti-CD81, Alexa Fluor 647, magenta) in co-culture with 2° 293T DsRed actin (red) expressing cells. Right hand panels lack the DsRed layer for improved visual clarity of otherwise identical frames. Genomes co-localized with tetraspanin are white (arrows, boxes). Genomes co-localized with DsRed actin are yellow. Deconvolution microscopy was performed on live cells by collecting series of z -stacks (0.5 µm) every minute for 10 minutes, elapsed time is indicated in black.

    Article Snippet: For pronase washes, cells were pelleted immediately following transduction and resuspended in 1 mg/ml pronase (Roche) for 10 minutes at 37°C followed by two washes in media containing 10% FBS.

    Techniques: Plasmid Preparation, Staining, Live Cell Imaging, Labeling, Co-Culture Assay, Expressing, Microscopy

    Endosomal acidification increases the proportional efficiency of secondary transfer. (A) Effect of endosomal acidification on 2° transfer. SupT1 carrier cells were pretreated with escalating doses of ammonium chloride or chloroquine (B) followed by vector exposure, pronase wash, and 24-hour coculture with 293T cells. Primary transduction (gray), secondary transfer (black), and proportional % efficiency of secondary transfer (red) are shown. (C,D) Colocalization of vector genomes with representative endosomal markers. Jurkat cells and (E,F) SupT1 cells were exposed to GFP-vpr tagged vector for 1 (C,E) or 24 hours (D,E). Cells were stained with antibodies against indicated endosomal cellular compartments ( x-axis ), as well as Golgin 97 (negative control) and LAMP1 (positive control), and cells were visualized by immunofluorescent microscopy for determination of co-localization of particles and specific compartment markers. The total number of particles (gray, numerator in each column) and total number of co-localized particles (black, denominator in each column) were counted.

    Journal: PLoS ONE

    Article Title: Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells

    doi: 10.1371/journal.pone.0006219

    Figure Lengend Snippet: Endosomal acidification increases the proportional efficiency of secondary transfer. (A) Effect of endosomal acidification on 2° transfer. SupT1 carrier cells were pretreated with escalating doses of ammonium chloride or chloroquine (B) followed by vector exposure, pronase wash, and 24-hour coculture with 293T cells. Primary transduction (gray), secondary transfer (black), and proportional % efficiency of secondary transfer (red) are shown. (C,D) Colocalization of vector genomes with representative endosomal markers. Jurkat cells and (E,F) SupT1 cells were exposed to GFP-vpr tagged vector for 1 (C,E) or 24 hours (D,E). Cells were stained with antibodies against indicated endosomal cellular compartments ( x-axis ), as well as Golgin 97 (negative control) and LAMP1 (positive control), and cells were visualized by immunofluorescent microscopy for determination of co-localization of particles and specific compartment markers. The total number of particles (gray, numerator in each column) and total number of co-localized particles (black, denominator in each column) were counted.

    Article Snippet: For pronase washes, cells were pelleted immediately following transduction and resuspended in 1 mg/ml pronase (Roche) for 10 minutes at 37°C followed by two washes in media containing 10% FBS.

    Techniques: Plasmid Preparation, Transduction, Staining, Negative Control, Positive Control, Microscopy

    Cultures of hepatic stellate cells isolated via iohexol density gradient centrifugation without or with subsequent cell sorting. Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol. Cells were cultured directly after the gradient for one day (a) and four days (b), where Kupffer cells (indicated by arrows) can be found in the HSC culture. Highly pure HSC after additional fluorescence-activated cell sorting (FACS) after one (c) and four days of culture are shown (d). Expression analysis of desmin (e) and phalloidin (f) of HSC after four days of culture, indicating proper maturation of HSC.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

    doi: 10.1155/2015/417023

    Figure Lengend Snippet: Cultures of hepatic stellate cells isolated via iohexol density gradient centrifugation without or with subsequent cell sorting. Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol. Cells were cultured directly after the gradient for one day (a) and four days (b), where Kupffer cells (indicated by arrows) can be found in the HSC culture. Highly pure HSC after additional fluorescence-activated cell sorting (FACS) after one (c) and four days of culture are shown (d). Expression analysis of desmin (e) and phalloidin (f) of HSC after four days of culture, indicating proper maturation of HSC.

    Article Snippet: Second, perfusion buffer 2 (8 g/L NaCl, 400 mg/L KCl, 78 mg/L NaH2 PO4 × H2 O, 151 mg/L NaHPO4 × 2 H2 O, 2380 mg/L HEPES, 350 mg/L NaHCO3 , 560 mg/L CaCl2 × 2 H2 O, and 6 mg/L phenol red, adjusted to pH 7.3–7.4 using 10 N NaOH, sterile filtered, and kept at 4°C until use) was applied for 4.5 minutes and supplemented with 0.5 mg/mL pronase E (Merck, Darmstadt, Germany).

    Techniques: Isolation, Gradient Centrifugation, FACS, Mouse Assay, Cell Culture, Fluorescence, Expressing

    Optimization of the isolation of primary hepatic stellate cells (HSC) based on iohexol density gradient centrifugation and fluorescence-activated cell sorting (FACS) (schematic depiction). In both strategies for cell purification, the mouse is anaesthetized before surgery, and the liver is then perfused via the Vena portae and drained through the Vena cava inferior using a two-step perfusion of the enzymes pronase and collagenase (a). Liver cells are harvested by gently tearing the liver into bits (b), followed by a postdigestion using a combination of both enzymes (c). The liver cells are subjected to iohexol density gradient centrifugation, after which HSC and Kupffer cells are located in the interphase between iohexol and buffer (d). The enriched HSC layer containing HSC, HSC-Kupffer cell doublets, and cellular debris can be used directly for cell culture of HSC (e) or can be cleared from HSC-Kupffer cell doublets and cellular debris using FACS based on the autofluorescence of retinol, using the UV laser of the cell sorter, resulting in highly pure HSC (f).

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

    doi: 10.1155/2015/417023

    Figure Lengend Snippet: Optimization of the isolation of primary hepatic stellate cells (HSC) based on iohexol density gradient centrifugation and fluorescence-activated cell sorting (FACS) (schematic depiction). In both strategies for cell purification, the mouse is anaesthetized before surgery, and the liver is then perfused via the Vena portae and drained through the Vena cava inferior using a two-step perfusion of the enzymes pronase and collagenase (a). Liver cells are harvested by gently tearing the liver into bits (b), followed by a postdigestion using a combination of both enzymes (c). The liver cells are subjected to iohexol density gradient centrifugation, after which HSC and Kupffer cells are located in the interphase between iohexol and buffer (d). The enriched HSC layer containing HSC, HSC-Kupffer cell doublets, and cellular debris can be used directly for cell culture of HSC (e) or can be cleared from HSC-Kupffer cell doublets and cellular debris using FACS based on the autofluorescence of retinol, using the UV laser of the cell sorter, resulting in highly pure HSC (f).

    Article Snippet: Second, perfusion buffer 2 (8 g/L NaCl, 400 mg/L KCl, 78 mg/L NaH2 PO4 × H2 O, 151 mg/L NaHPO4 × 2 H2 O, 2380 mg/L HEPES, 350 mg/L NaHCO3 , 560 mg/L CaCl2 × 2 H2 O, and 6 mg/L phenol red, adjusted to pH 7.3–7.4 using 10 N NaOH, sterile filtered, and kept at 4°C until use) was applied for 4.5 minutes and supplemented with 0.5 mg/mL pronase E (Merck, Darmstadt, Germany).

    Techniques: Isolation, Gradient Centrifugation, Fluorescence, FACS, Purification, Cell Culture

    Hepatic stellate cell functionality in vitro . Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol and fluorescence-activated cell sorting (FACS). The HSC were cultured in DMEM with 10% fetal calf serum, and some plates were stimulated with lipopolysaccharides (LPS) at 100 ng/mL (after five days of culture) for another 48 hours. Changes in the cell number during culture were determined from time lapse microscopy (a) and statistical summary (b). HSC were cultured for five days on tissue culture-treated polystyrene in DMEM with 10% fetal calf serum including culture inserts for self-insertion (“scratch assay”). To start horizontal migration, the plastic inserts were removed and HSC migrated (c) and were quantified using software (d). HSC were cultured for designated periods and quantitative real-time PCR was performed to study the expression of α smooth muscle actin ( α SMA), collagen 1 (Col1A1), or the transforming growth factor β (TGF β ) as markers of HSC activation. Gene expression was normalized to β -actin expression of cells that were lysed directly after isolation at day zero (e). Mean ± SD of three independent experiments.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

    doi: 10.1155/2015/417023

    Figure Lengend Snippet: Hepatic stellate cell functionality in vitro . Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol and fluorescence-activated cell sorting (FACS). The HSC were cultured in DMEM with 10% fetal calf serum, and some plates were stimulated with lipopolysaccharides (LPS) at 100 ng/mL (after five days of culture) for another 48 hours. Changes in the cell number during culture were determined from time lapse microscopy (a) and statistical summary (b). HSC were cultured for five days on tissue culture-treated polystyrene in DMEM with 10% fetal calf serum including culture inserts for self-insertion (“scratch assay”). To start horizontal migration, the plastic inserts were removed and HSC migrated (c) and were quantified using software (d). HSC were cultured for designated periods and quantitative real-time PCR was performed to study the expression of α smooth muscle actin ( α SMA), collagen 1 (Col1A1), or the transforming growth factor β (TGF β ) as markers of HSC activation. Gene expression was normalized to β -actin expression of cells that were lysed directly after isolation at day zero (e). Mean ± SD of three independent experiments.

    Article Snippet: Second, perfusion buffer 2 (8 g/L NaCl, 400 mg/L KCl, 78 mg/L NaH2 PO4 × H2 O, 151 mg/L NaHPO4 × 2 H2 O, 2380 mg/L HEPES, 350 mg/L NaHCO3 , 560 mg/L CaCl2 × 2 H2 O, and 6 mg/L phenol red, adjusted to pH 7.3–7.4 using 10 N NaOH, sterile filtered, and kept at 4°C until use) was applied for 4.5 minutes and supplemented with 0.5 mg/mL pronase E (Merck, Darmstadt, Germany).

    Techniques: In Vitro, Isolation, Mouse Assay, Gradient Centrifugation, Fluorescence, FACS, Cell Culture, Time-lapse Microscopy, Migration, Software, Real-time Polymerase Chain Reaction, Expressing, Activation Assay