آزمون سمیت ژنتیکی | Comet Assay

آزمون سمیت ژنتیکی کامت جهت بررسی آسیب DNA سلولی ناشی از تماس با ماده آزمایشی انجام می شود. در این روش سلول ها تحت تیمار با ماده آزمایشی قرار می گیرند و سپس آسیب DNA با استفاده از بررسی پارامتر های طول Comet Tails،  درصد محتویات Tailed DNA، Tail Moment و Olive Tail Moment توسط نرم افزار Opencomet مورد ارزیابی قرار می گیرد.

#آزمون سمیت ژنتیکی#آزمون سمیت ژنتیکی به روش کامت اسی#تست سمیت ژنتیکی به روش کامت اسی#آزمون سمیت ژنی به روش کامت اسی#تست سمیت ژنی به روش کامت اسی#آزمون سمیت ژنی کامت اسی#تست سمیت ژنی کامت اسی#Hcl,k sldj Ckd ;hlj hsd#jsj sldj Ckjd;d ;hlj hsd#آزمون سمیت ژنی comet assay#تست سمیت ژنتیکی comey assay#تست سمیت ژنی comet#تست سمیت ژنتیکی comet

روش کار

• جهت انجام آزمون سمیت ژنی Comet Assay، ابتدا محلول های lysis solution، alkaline electrophoresis solution و  staining solution آماده خواهد شد.
• محلول یک درصد normal melting point agarose آماده و اسلاید شیشه ای داخل ژل غوطه ور خواهد شد تا یک لایه سطح اسلاید را بپوشاند (Agarose-Coated Slides).
• اسلاید ها شماره گذاری شده و روی سطح صاف داخل یخچال قرار خواهند گرفت تا خشک شوند.
• لاین سلولی L-929 داخل محیط کشت DMEM کشت و پاساژ داده شده و به مدت ۲۴ ساعت تحت تیمار با نمونه آزمایشی یا عصاره استخراج شده از آن قرار خواهد گرفت.
• پس از ۲۴ ساعت، سلول ها تریپسینه و با استفاده از لام هموسایتومتر (نئوبار) از نظر زنده مانی بررسی و غلظت سلولی  2×10⁵ cells/ml آماده خواهد شد.
• در کنار نمونه آزمایشی، نمونه شاهد منفی و شاهد مثبت جهت مقایسه آماری در نظر گرفته خواهد شد.
• محلول یک درصد low melting point agarose حاوی سوسپانسیون سلولی آماده و حجم ثابتی از آن بر روی اسلاید ریخته خواهد شد.
• اسلاید ها به مدت ۲۴ ساعت داخل محلول lysis solution قرار خواهند گرفت.

در طول پروسه لیز سلولی، pH محلول بالای 12.3 حفظ خواهد شد تا از ترمیم DNA و خطا در آزمون جلوگیری شود.

• اسلاید ها به آرامی از محلول lysis solution خارج و بافر اضافی از اسلاید پاک خواهند شد.
• اسلاید ها به مدت یک ساعت داخل محلول alkaline electrophoresis solution قرار خواهند گرفت.
• اسلاید ها داخل تانک الکتروفورز حاوی محلول alkaline electrophoresis solution قرار گرفته و به مدت ۳۰ دقیقه الکتروفورز خواهند شد.
• اسلاید ها از تانک الکتروفورز خارج و محلول اضافی از روی لام ها پاک خواهد شد.
• اسلاید ها به مدت 10 تا 15 دقیقه در دمای اتاق و فضای تاریک قرار گرفته تا خشک شوند.
• اسلاید ها به مدت 15 دقیقه تحت تیمار با 50 تا 100 میکرولیتر از محلول رنگ safe stain قرار خواهند گرفت.
• حداقل 50 سلول از هر اسلاید زیر میکرویکوپ فلورسنت با بزرگنمایی 10X، از نظر شکست DNA بررسی خواهد شد و در نهایت توسط نرم افزار  Opencomet از نظر پارامترهای طول Comet Tails،  درصد محتویات Tailed DNA، Tail Moment و Olive Tail Moment به صورت کمی مورد بررسی قرار خواهند گرفت.

جهت نگهداری طولانی مدت، اسلاید داخل الکل ۷۰ درصد غوطه ور خواهد شد و جهت حذف بک گراند فلورسنت، پیش از مشاهده اسلاید زیر میکروسکوپ فلورسنت، یک لایه نازک از ژل روی لام ریخته خواهد شد.

#آزمون سمیت ژنتیکی#آزمون سمیت ژنتیکی به روش کامت اسی#تست سمیت ژنتیکی به روش کامت اسی#آزمون سمیت ژنی به روش کامت اسی#تست سمیت ژنی به روش کامت اسی#آزمون سمیت ژنی کامت اسی#تست سمیت ژنی کامت اسی#Hcl,k sldj Ckd ;hlj hsd#jsj sldj Ckjd;d hlj hsd#آزمون سمیت ژنی comet assay#تست سمیت ژنتیکی comey assay#تست سمیت ژنی comet#تست سمیت ژنتیکی comet

استاندارد های زیست سازگاری | References

ISO 10993-1: Biological Evaluation of Medical Devices – Evaluation and testing within a risk management process.

ISO 10993-2: Biological Evaluation of Medical Devices – Animal welfare requirements.

ISO 10993-3: Biological Evaluation of Medical Devices – Tests for genotoxicity, carcinogenicity and reproductive toxicity.

ISO 10993-4: Biological Evaluation of Medical Devices – Selection of tests for interactions with blood.

ISO 10993-5: Biological Evaluation of Medical Devices – Tests for in vitro cytotoxicity.

ISO 10993-6: Biological Evaluation of Medical Devices – Tests for local effects after implantation.

ISO 10993-7: Biological Evaluation of Medical Devices – Ethylene oxide sterilization residuals.

ISO 10993-8: Biological Evaluation of Medical Devices – Selection and qualification of reference materials for biological tests.

ISO 10993-9: Biological Evaluation of Medical Devices – Framework for identification and quantification of potential degradation products.

ISO 10993-10: Biological Evaluation of Medical Devices – Tests for skin sensitization.

ISO 10993-11: Biological Evaluation of Medical Devices – Tests for systemic toxicity.

ISO 10993-12: Biological Evaluation of Medical Devices – Sample preparation and reference materials.

ISO 10993-13: Biological Evaluation of Medical Devices – Identification and quantification of degradation products from polymeric medical devices.

ISO 10993-14: Biological Evaluation of Medical Devices – Identification and quantification of degradation products from ceramics.

ISO 10993-15: Biological Evaluation of Medical Devices – Identification and quantification of degradation products from metals and alloys.

ISO 10993-16: Biological Evaluation of Medical Devices – Toxicokinetic study design for degradation products and leachables.

ISO 10993-17: Biological Evaluation of Medical Devices – Toxicological risk assessment of medical device constituents.

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ISO 10993-19: Biological Evaluation of Medical Devices – Physico-chemical, morphological and topographical characterization of materials.

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OECD Test No. 494: Guidelines for the Testing of Chemicals – Vitrigel-Eye Irritancy Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage.

OECD Test No. 495: Guidelines for the Testing of Chemicals – Ros (Reactive Oxygen Species) Assay for Photoreactivity.

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OECD Test No. 497: Guidelines for the Testing of Chemicals – Defined Approaches on Skin Sensitisation.

OECD Test No. 498: Guidelines for the Testing of Chemicals – In vitro Phototoxicity – Reconstructed Human Epidermis Phototoxicity test method.

OECD Test No. 19: Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assessment.

#آزمون سمیت ژنتیکی#آزمون سمیت ژنتیکی به روش کامت اسی#تست سمیت ژنتیکی به روش کامت اسی#آزمون سمیت ژنی به روش کامت اسی#تست سمیت ژنی به روش کامت اسی#آزمون سمیت ژنی کامت اسی#تست سمیت ژنی کامت اسی#Hcl,k sldj Ckd ;hlj hsd#jsj sldj Ckjd;d ;hlj hsd#آزمون سمیت ژنی comet assay#تست سمیت ژنتیکی comey assay#تست سمیت ژنی comet#تست سمیت ژنتیکی comet