Red Blood Cell Lysis:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Filtration:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Purification:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Article Title: Diabetes exacerbates destructive inflammation by activating the CD137L-CD137 axis in dendritic and IL-17 + T cells
Article Snippet: .. Spleen was collected from 6-10 weeks old C57BL/6 mice, minced, strained (VWR, cat#76327100), and RBC was lysed using Ammonium-Chloride-Potassium (ACK) buffer (Quality Biologicals, cat#118156101). γδ T-cells were purified by positive selection with magnetic beads per manufacturer’s protocol (Cat# 130-092-125, Miltenyi Biotec, Germany). .. Cells were 10 resuspended into RPMI 1640 containing 10% FBS, recombinant mouse IL-2 (5ng/ml) (R&D systems, cat# 402-ML-020/CF) and activated using plate-bound anti-CD3 (1ug/ml) (Invivo, cat#BE0002) and anti-CD28 (5ug/ml) (Invivo, cat#BE0015-5).
Single Cell:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Suspension:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Magnetic Cell Separation:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Cell Isolation:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Article Title: Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction
Article Snippet: .. Spleen cells were resuspended in erythrocyte lysis buffer (150 mM Ammonium chloride NH4Cl, 10 mM Potassium bicarbonate KHCO3, and 0.1 mM EDTA in distilled water) for 10min, then washed. γδ T cells were isolated with mouse TCRγ/δ T Cell Isolation Kit (Miltenyi Biotec, 130-092-125) according to the manufacturer instructions. .. The purity was checked by flow cytometry analysis. γδ T cells were either used directly for further experiments in a non-stimulated state or stimulated and amplified for 5 days in complete medium (RPMI Glutamax, 10%FCS, 1%P/S, 10μM Betamercaptoethanol, 10mM HEPES, 1mM Na Pyruvate, 100μM non-essential amino acids) together with anti-CD3 (Thermofisher, 16-0032-85, 2μg/mL), anti-CD28 (Thermofisher, 16-0281-85, 2μg/ml) and Il-2 (PeproTech®, 212-12, 10ng/mL).
Flow Cytometry:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Cell Culture:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
In Vitro:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Incubation:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Enzyme-linked Immunosorbent Assay:Article Title: γδ T cell-stromal networks modulate matrix composition and vascularity in foreign body response.
Article Snippet: .. Red blood cell lysis was performed with 1x Pharm Lyse Lysing Buffer (BD 555899) and followed by filtration through 40 μm cell strainers (Falcon 352340). γδ T cells were purified from the single-cell suspension using MACS with the mouse TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec 130-092-125) as per manufacturer protocol. γδ T cell purity was confirmed using flow cytometry. γδ T cells were cultured in T cell media consisting of RPMI-1640 supplemented with 10% heatinactivated FBS, 1% Pen-Strep, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids (Gibco 11140-050), and 1% HEPES. γδ T cells were seeded at 200,000 – 300,000 cells/well in a 96-well TC-treated flat bottom plate in T cell media and skewed in vitro using plate-bound anti-CD3 antibody (10 μg/mL) (BioLegend 100238) and either IL2 (10 ng/mL) (BioLegend 575402) for γδIFNγ or IL1β (BioLegend 575102), IL7 (BioLegend 577802), IL21 (BioLegend 574502), and IL23 (10 ng/mL each) (BioLegend 589002) for γδ17 (adapted from 93,245,246). γδ T cells were incubated in respective cytokine cocktail at 37C with 5% CO2 for 72 hours and phenotypic skewing was confirmed via enzyme-linked immunosorbent assay (ELISA) for IFNγ (Invitrogen 88-7314) and IL17A homodimer (Invitrogen 88-7371) on supernatant as per manufacturer’s protocol. .. Sample Preparation: VML-injured muscles with fibrotic PCL implants were carefully excised and placed into RNAlater Stabilization Solution (Invitrogen AM7024) for at least 48 hours at 4C, then transferred into cold TRIzol Reagent (Invitrogen 15596018).
Isolation:
Article Title: Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction
Article Snippet: .. Spleen cells were resuspended in erythrocyte lysis buffer (150 mM Ammonium chloride NH4Cl, 10 mM Potassium bicarbonate KHCO3, and 0.1 mM EDTA in distilled water) for 10min, then washed. γδ T cells were isolated with mouse TCRγ/δ T Cell Isolation Kit (Miltenyi Biotec, 130-092-125) according to the manufacturer instructions. .. The purity was checked by flow cytometry analysis. γδ T cells were either used directly for further experiments in a non-stimulated state or stimulated and amplified for 5 days in complete medium (RPMI Glutamax, 10%FCS, 1%P/S, 10μM Betamercaptoethanol, 10mM HEPES, 1mM Na Pyruvate, 100μM non-essential amino acids) together with anti-CD3 (Thermofisher, 16-0032-85, 2μg/mL), anti-CD28 (Thermofisher, 16-0281-85, 2μg/ml) and Il-2 (PeproTech®, 212-12, 10ng/mL).
Article Title: Homotypic SLAMF1:SLAMF1 interactions between innate T cells and neutrophils activate fungal killing by neutrophils
Article Snippet: .. CD4 + and TCRγδ + T cells were isolated from the lungs of infected mice (16 hours post-challenge) following lung processing protocol referenced above to harvest leukocytes, then according to the manufacture’s isolation protocols (Miltenyi Isolation kits (CD4+T cells; Cat#130-104-454 and TCRγδ; Cat# 130-092-125). ..
Lysis:
Article Title: Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction
Article Snippet: .. Spleen cells were resuspended in erythrocyte lysis buffer (150 mM Ammonium chloride NH4Cl, 10 mM Potassium bicarbonate KHCO3, and 0.1 mM EDTA in distilled water) for 10min, then washed. γδ T cells were isolated with mouse TCRγ/δ T Cell Isolation Kit (Miltenyi Biotec, 130-092-125) according to the manufacturer instructions. .. The purity was checked by flow cytometry analysis. γδ T cells were either used directly for further experiments in a non-stimulated state or stimulated and amplified for 5 days in complete medium (RPMI Glutamax, 10%FCS, 1%P/S, 10μM Betamercaptoethanol, 10mM HEPES, 1mM Na Pyruvate, 100μM non-essential amino acids) together with anti-CD3 (Thermofisher, 16-0032-85, 2μg/mL), anti-CD28 (Thermofisher, 16-0281-85, 2μg/ml) and Il-2 (PeproTech®, 212-12, 10ng/mL).
Selection:Article Title: Diabetes exacerbates destructive inflammation by activating the CD137L-CD137 axis in dendritic and IL-17 + T cells
Article Snippet: .. Spleen was collected from 6-10 weeks old C57BL/6 mice, minced, strained (VWR, cat#76327100), and RBC was lysed using Ammonium-Chloride-Potassium (ACK) buffer (Quality Biologicals, cat#118156101). γδ T-cells were purified by positive selection with magnetic beads per manufacturer’s protocol (Cat# 130-092-125, Miltenyi Biotec, Germany). .. Cells were 10 resuspended into RPMI 1640 containing 10% FBS, recombinant mouse IL-2 (5ng/ml) (R&D systems, cat# 402-ML-020/CF) and activated using plate-bound anti-CD3 (1ug/ml) (Invivo, cat#BE0002) and anti-CD28 (5ug/ml) (Invivo, cat#BE0015-5).
Magnetic Beads:Article Title: Diabetes exacerbates destructive inflammation by activating the CD137L-CD137 axis in dendritic and IL-17 + T cells
Article Snippet: .. Spleen was collected from 6-10 weeks old C57BL/6 mice, minced, strained (VWR, cat#76327100), and RBC was lysed using Ammonium-Chloride-Potassium (ACK) buffer (Quality Biologicals, cat#118156101). γδ T-cells were purified by positive selection with magnetic beads per manufacturer’s protocol (Cat# 130-092-125, Miltenyi Biotec, Germany). .. Cells were 10 resuspended into RPMI 1640 containing 10% FBS, recombinant mouse IL-2 (5ng/ml) (R&D systems, cat# 402-ML-020/CF) and activated using plate-bound anti-CD3 (1ug/ml) (Invivo, cat#BE0002) and anti-CD28 (5ug/ml) (Invivo, cat#BE0015-5).
Infection:Article Title: Homotypic SLAMF1:SLAMF1 interactions between innate T cells and neutrophils activate fungal killing by neutrophils
Article Snippet: .. CD4 + and TCRγδ + T cells were isolated from the lungs of infected mice (16 hours post-challenge) following lung processing protocol referenced above to harvest leukocytes, then according to the manufacture’s isolation protocols (Miltenyi Isolation kits (CD4+T cells; Cat#130-104-454 and TCRγδ; Cat# 130-092-125). ..
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