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tau (k18) p301l mutant pre-formed fibrils  (StressMarq)


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    Structured Review

    StressMarq tau (k18) p301l mutant pre-formed fibrils
    Tau (K18) P301l Mutant Pre Formed Fibrils, supplied by StressMarq, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/SPR-330/Tau+(K18)+P301L+Mutant+Pre-formed+Fibrils/custom%40spr-330%4040301692
    Average 93 stars, based on 5 article reviews
    tau (k18) p301l mutant pre-formed fibrils - by Bioz Stars, 2026-09
    93/100 stars

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    Related Articles

    Stable Transfection:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Generated:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Gentle:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Saline:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Sterility:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    SPR Assay:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Article Title: Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils.
    Article Snippet: Immunoblots shown in Fig. S17 were prepared by blotting tau and α-synuclein proteins onto nitrocellulose 23 membrane (GE Healthcare Life Science, 10600004). .. Human HT40 fibrils (Stressmarq, SPR-329), human 0N3R 24 fibrils (Stressmarq, SPR-491), human K18 P301L fibrils (Stressmaq, SPR-330), mouse 2N4R fibrils (Stressmarq, 25 SPR-475), human α-synuclein fibrils (Stressmarq, SPR-317), human N-acetylated α-synuclein fibrils (Stressmarq, 26 SPR-332), and mouse α-synuclein fibrils (Stressmarq, SPR-324) were first sonicated for 5 min (30 s on, 30 s off) 1 before loading onto the nitrocellulose membrane. .. Human α-synuclein oligomers (Stressmarq, SPR-484) were 2 loaded onto the nitrocellulose membrane without sonication.

    Article Title: Facile generation of drug-like conformational antibodies specific for amyloid fibrils.
    Article Snippet: Antibodies that recognize insoluble antigens, such as amyloid fibrils associated with neurodegenerative disorders, are important for research, diagnostic and therapeutic applications.. However, these types of antibodies are difficult to generate, typically require animal immunization and also commonly require humanization in the case of therapeutic applications.. Here we report a methodology for generating high-quality, fully human, conformation-specific antibodies against amyloid fibrils using a published human nonimmune library, yeast-surface display and quantitative fluorescence-activated cell sorting.

    Electron Microscopy:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Blocking Assay:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Cell Culture:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Transduction:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Hood:

    Article Title: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.
    Article Snippet: .. Materials Geltrex (see recipe) DMEM/F12 (e.g., Life Technologies, cat. no. 11320033) TesR-E8 medium (see recipe) 10 μg/ml NT3 (see recipe) 20 μg/ml BDNF (see recipe) 40 mg/ml doxycycline (see recipe) RI (see recipe) Near-confluent plate of human iPSCs with stably integrated doxycycline-inducible reverse tetracycline (rtTA)/Ngn2 (see Support Protocol 1; generated as described in Frega et al., 2017) or EBiSC line BIOni010-C-13 (see Support Protocol 1 and Schmid et al., 2021; e.g., European Collection of Authenticated Cell Cultures, cat. no. 66540561) Gentle cell dissociation reagent (e.g., StemCell Technologies, cat. no. 07174) Trypan blue (e.g., Life Technologies, cat. no. 15250061) DMEM (see recipes) Viral particles encoding fluorescently tagged 2N4R human tau (microtubule-associated protein tau [MAPT]) containing the P301L (FTDtau1) or P301L+S320F (FTDtau1+2) mutations (prepared as described in Moro et al., 2021; Naldini et al., 1996) Accutase (e.g., Sigma-Aldrich, cat. no. SF006) Neurobasal medium with or without FBS (see recipes) Confluent plate of human primary astrocytes (see Support Protocol 2; e.g., ScienCell, cat. no. 1800) Phosphate-buffered saline (PBS), sterile or filtered (see recipe) 4 mM AraC (see recipe) Tau PFFs (e.g., Stressmarq, cat. no. SPR-330, or prepared as described in Peeraer et al., 2015; Wiersma et al., 2019) 0.1 M sodium acetate (e.g., Sigma-Aldrich, cat. no. 32319) 4% (w/v) paraformaldehyde (PFA; e.g., Electron Microscopy Sciences, cat. no. 15710-S) Methanol (MeOH), ice-cold (e.g., Millipore Sigma, cat. no. 106012) Blocking buffer (see recipe) Primary antibodies (see Table 1) Alexa Fluor–conjugated secondary antibodies, 1:1000 dilution (e.g., Invitrogen) 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; see recipe) 96-well plates, black (e.g., Greiner, cat. no. 655090) 37°C, 20% O2, 5% CO2 cell culture incubator Parafilm M 1.5-ml microcentrifuge tube (e.g., Eppendorf, cat. no. 0030108051) 15-ml conical tubes Tabletop centrifuge with rotor equipped for 1.5- and 15-ml tubes Hemocytometer (e.g., Bürker chamber; NanoEntek, cat. no. DHC-B01) Aluminum foil Probe sonicator CAUTION: Viral transduction should be carried out in a class 2 biosafety cabinet to avoid viral contamination. ..

    Sonication:

    Article Title: Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils.
    Article Snippet: Immunoblots shown in Fig. S17 were prepared by blotting tau and α-synuclein proteins onto nitrocellulose 23 membrane (GE Healthcare Life Science, 10600004). .. Human HT40 fibrils (Stressmarq, SPR-329), human 0N3R 24 fibrils (Stressmarq, SPR-491), human K18 P301L fibrils (Stressmaq, SPR-330), mouse 2N4R fibrils (Stressmarq, 25 SPR-475), human α-synuclein fibrils (Stressmarq, SPR-317), human N-acetylated α-synuclein fibrils (Stressmarq, 26 SPR-332), and mouse α-synuclein fibrils (Stressmarq, SPR-324) were first sonicated for 5 min (30 s on, 30 s off) 1 before loading onto the nitrocellulose membrane. .. Human α-synuclein oligomers (Stressmarq, SPR-484) were 2 loaded onto the nitrocellulose membrane without sonication.

    Article Title: Facile generation of drug-like conformational antibodies specific for amyloid fibrils.
    Article Snippet: Antibodies that recognize insoluble antigens, such as amyloid fibrils associated with neurodegenerative disorders, are important for research, diagnostic and therapeutic applications.. However, these types of antibodies are difficult to generate, typically require animal immunization and also commonly require humanization in the case of therapeutic applications.. Here we report a methodology for generating high-quality, fully human, conformation-specific antibodies against amyloid fibrils using a published human nonimmune library, yeast-surface display and quantitative fluorescence-activated cell sorting.

    Membrane:

    Article Title: Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils.
    Article Snippet: Immunoblots shown in Fig. S17 were prepared by blotting tau and α-synuclein proteins onto nitrocellulose 23 membrane (GE Healthcare Life Science, 10600004). .. Human HT40 fibrils (Stressmarq, SPR-329), human 0N3R 24 fibrils (Stressmarq, SPR-491), human K18 P301L fibrils (Stressmaq, SPR-330), mouse 2N4R fibrils (Stressmarq, 25 SPR-475), human α-synuclein fibrils (Stressmarq, SPR-317), human N-acetylated α-synuclein fibrils (Stressmarq, 26 SPR-332), and mouse α-synuclein fibrils (Stressmarq, SPR-324) were first sonicated for 5 min (30 s on, 30 s off) 1 before loading onto the nitrocellulose membrane. .. Human α-synuclein oligomers (Stressmarq, SPR-484) were 2 loaded onto the nitrocellulose membrane without sonication.

    Article Title: Facile generation of drug-like conformational antibodies specific for amyloid fibrils.
    Article Snippet: Antibodies that recognize insoluble antigens, such as amyloid fibrils associated with neurodegenerative disorders, are important for research, diagnostic and therapeutic applications.. However, these types of antibodies are difficult to generate, typically require animal immunization and also commonly require humanization in the case of therapeutic applications.. Here we report a methodology for generating high-quality, fully human, conformation-specific antibodies against amyloid fibrils using a published human nonimmune library, yeast-surface display and quantitative fluorescence-activated cell sorting.



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