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Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
Dispase, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane"

Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane

Journal: Toxicology in vitro : an international journal published in association with BIBRA

doi: 10.1016/j.tiv.2017.03.002

Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using dispase to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
Figure Legend Snippet: Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using dispase to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.

Techniques Used: Glo Assay, Western Blot, Positive Control, Isolation, Standard Deviation

Related Articles

Isolation:

Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane
Article Snippet: .. For HBMECs grown on Matrigel®, cells were isolated by aspirating normal growth media and adding equivalent culture volumes of Dispase (Corning), incubating at 37 °C for 30 min, then washing 2x with ice cold 1xPBS prior to lysis. .. Protein concentration was determined for each sample using a Bicinchoninic acid (BCA) protein assay kit (Thermo-Fisher/Pierce).

Incubation:

Article Title: Dlk1-Mediated Temporal Regulation of Notch Signaling Is Required for Differentiation of Alveolar Type II to Type I Cells during Repair
Article Snippet: .. The lungs were removed and incubated in dispase (Corning) at room temperature for 45 minutes. ..

Article Title: SARS-CoV-2 Productively Infects Human Gut Enterocytes
Article Snippet: .. Bronchial tissues of adult lungs were cut into ~4mm sections, washed in Advanced DMEM supplemented with GlutaMax (Thermo Fisher), HEPES, penicillin (10,000 IU/mL) and streptomycin (10,000 IU/mL) (AdDF+++), and incubated with dispase (Corning) mixed with airway organoid (AO) medium ( ) at a 1:1 ratio for 1 hour at 37°C. .. Isolation of human bronchial airway stem cells was performed using a protocol adapted from Sachs and colleagues (2019).

Dissection:

Article Title: Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection
Article Snippet: .. Briefly, perfused lungs treated with dispase (Corning, 47743-724) and DNase (50 mg/mL; Sigma-Aldrich, D4513-1VL) were subjected to manual dissection. .. Single-cell suspensions were enriched for epithelial cells using anti-EpCAM magnetic microbeads (Miltenyi Biotec, 130-105-958).

Lysis:

Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane
Article Snippet: .. For HBMECs grown on Matrigel®, cells were isolated by aspirating normal growth media and adding equivalent culture volumes of Dispase (Corning), incubating at 37 °C for 30 min, then washing 2x with ice cold 1xPBS prior to lysis. .. Protein concentration was determined for each sample using a Bicinchoninic acid (BCA) protein assay kit (Thermo-Fisher/Pierce).

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  • 90
    Corning Life Sciences dispase solution
    Differentiation of HFKs Expanded with the EpiX Medium (A) Addition of 1 mM CaCl 2 to the EpiX medium induced the HFKs to differentiate in 24 hr. (B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl 2 for 7 days. ZO-1, zonula occludens-1. (C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl 2 (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl 2 . (E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl 2 for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in <t>dispase</t> at 37°C. (F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium. (G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis. (H) Most cells in the basal layer were stained positive for the proliferation marker Ki67. (I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).
    Dispase Solution, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dispase solution - by Bioz Stars, 2021-01
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    88
    Corning Life Sciences dispase mechanical dissociation
    ID6 porcine iPSC colonies formed following 2 weeks of cryopreservation. Colonies were broken into uniform-size clumps (see Fig. 5B ) by mechanical dissociation after <t>dispase</t> treatment and then cryopreserved. ( A ), LN 2 without Ficoll 70; ( B ), −80 °C without Ficoll 70; ( C ), −80 °C with Ficoll 70. Images were taken at d 4 after thawing.
    Dispase Mechanical Dissociation, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    dispase mechanical dissociation - by Bioz Stars, 2021-01
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    Image Search Results


    Differentiation of HFKs Expanded with the EpiX Medium (A) Addition of 1 mM CaCl 2 to the EpiX medium induced the HFKs to differentiate in 24 hr. (B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl 2 for 7 days. ZO-1, zonula occludens-1. (C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl 2 (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl 2 . (E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl 2 for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in dispase at 37°C. (F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium. (G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis. (H) Most cells in the basal layer were stained positive for the proliferation marker Ki67. (I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).

    Journal: Cell reports

    Article Title: Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF-β Signaling

    doi: 10.1016/j.celrep.2018.09.072

    Figure Lengend Snippet: Differentiation of HFKs Expanded with the EpiX Medium (A) Addition of 1 mM CaCl 2 to the EpiX medium induced the HFKs to differentiate in 24 hr. (B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl 2 for 7 days. ZO-1, zonula occludens-1. (C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl 2 (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl 2 . (E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl 2 for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in dispase at 37°C. (F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium. (G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis. (H) Most cells in the basal layer were stained positive for the proliferation marker Ki67. (I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).

    Article Snippet: Keratinocytes isolation and expansion Fresh human skin purchased from ZenBio Inc. was cut into small pieces and placed in Dispase solution (Corning) at 4C overnight.

    Techniques: Immunofluorescence, Staining, Cell Culture, Incubation, Injection, Mouse Assay, Marker

    Differentiation of HFKs Expanded with the EpiX Medium (A) Addition of 1 mM CaCl 2 to the EpiX medium induced the HFKs to differentiate in 24 hr. (B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl 2 for 7 days. ZO-1, zonula occludens-1. (C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl 2 (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl 2 . (E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl 2 for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in dispase at 37°C. (F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium. (G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis. (H) Most cells in the basal layer were stained positive for the proliferation marker Ki67. (I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).

    Journal: Cell reports

    Article Title: Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF-β Signaling

    doi: 10.1016/j.celrep.2018.09.072

    Figure Lengend Snippet: Differentiation of HFKs Expanded with the EpiX Medium (A) Addition of 1 mM CaCl 2 to the EpiX medium induced the HFKs to differentiate in 24 hr. (B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl 2 for 7 days. ZO-1, zonula occludens-1. (C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl 2 (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl 2 . (E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl 2 for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in dispase at 37°C. (F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium. (G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis. (H) Most cells in the basal layer were stained positive for the proliferation marker Ki67. (I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).

    Article Snippet: Fresh human skin purchased from ZenBio Inc. was cut into small pieces and placed in Dispase solution (Corning) at 4C overnight.

    Techniques: Immunofluorescence, Staining, Cell Culture, Incubation, Injection, Mouse Assay, Marker

    ID6 porcine iPSC colonies formed following 2 weeks of cryopreservation. Colonies were broken into uniform-size clumps (see Fig. 5B ) by mechanical dissociation after dispase treatment and then cryopreserved. ( A ), LN 2 without Ficoll 70; ( B ), −80 °C without Ficoll 70; ( C ), −80 °C with Ficoll 70. Images were taken at d 4 after thawing.

    Journal: Scientific Reports

    Article Title: Efficient long-term cryopreservation of pluripotent stem cells at −80 °C

    doi: 10.1038/srep34476

    Figure Lengend Snippet: ID6 porcine iPSC colonies formed following 2 weeks of cryopreservation. Colonies were broken into uniform-size clumps (see Fig. 5B ) by mechanical dissociation after dispase treatment and then cryopreserved. ( A ), LN 2 without Ficoll 70; ( B ), −80 °C without Ficoll 70; ( C ), −80 °C with Ficoll 70. Images were taken at d 4 after thawing.

    Article Snippet: Spontaneous differentiation of hESC via embryoid body (EB) formation Colonies of hESC were dispersed by dispase/mechanical dissociation and transferred into EB differentiation medium, consisting of DMEM/F12, 15% FBS, 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM 2-mercaptoethanol, in low attachment plates (Corning).

    Techniques: