Comet Assay Slide: Platform a pụrụ ịdabere na ya maka nyocha mmebi DNA

Okwu
Komet assay, ihe nkiri ihe nkiri comet, slide DNA mebiri emebi, slide otu cell gel electrophoresis slide, ihe mkpuchi agarose, slide comet assay slide, alkaline comet assay, nnwale genotoxicity, nchọpụta nbibi DNA strand, Ntuziaka OECD 489

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Okwu mmalite
Nkenyocha nke comet, makwaara dị kaotu - cell gel electrophoresis nyocha, bụ usoro a na-ejikarị achọpụta nkwụsị DNA strand n'ogo otu-sel. Na Ntuziaka OECD 489, a kọwara nyocha ahụ dị ka usoro nke mkpụrụ ndụ ma ọ bụ nuclei na-etinye n'ime agarose na slides, lysed, wee tinye ya na elu - pH electrophoresis iji kpughee usoro njem DNA nke yiri comets.

Ihe slide comet assay karịrị elu nkwado dị mfe. Ọ bụ ikpo okwu na-ejide agarose-nlele agbakwunyere site na lysis, electrophoresis, ntụpọ, na onyonyo. N'okwu bara uru, ogo mmịfe nwere ike imetụta njide jel, ịdị n'otu onyonyo, na mmụgharị nke akara akara ala.

Kedu ihe bụ ihe mmịfe nke Comet Assay?

Ihe slide comet assay bụ ihe mmịfe microscope ma ọ bụ slide - dị ka mkpụrụ osisi emebere maka nkwadebe na nyocha ihe nyocha nke comet assay. Usoro nyocha nke nyocha na-agụnye iji agarose kpuchie slide ahụ, tinye mkpụrụ ndụ n'ime ala - agbaze-point agarose, wee hazie slide ahụ site na lysis na electrophoresis. Protocols rịba ama na enwere ike iji usoro mmịfe ọzọ na ihe nkiri hydrophilic, mana ọrụ bụ isi ka bụ otu: idowe akwa agarose kwụsiri ike n'oge nyocha.

N'ụkpụrụ ọkọlọtọ, a na-ebu ụzọ ekpuchi elu slide - na nkịtị-agbaze-point agarose iji melite nrapado nke oyi akwa sample. A na-ejikwa ihe mmịfe emechiri emechi n'azụmahịa iji mee ka nkwadebe dị mfe ma melite ngbanwe usoro ọrụ.

Ihe kpatara ịdị mma mmịfe ji dị mkpa

Nnwale comet nwere mmetụta, mana ọ na-achọkwa teknụzụ. Akwụkwọ akụkọ e bipụtara na-ekwu na nkwadebe nlele na nhazi slide na-arụ ọrụ - na-arụ ọrụ siri ike, na mmepụta dị ala ejedebela ogologo oge nyocha ahụ. Emepụtara ngbanwe dị elu n'otu akụkụ n'ihi na nhazi slide n'onwe ya nwere ike ịghọ ihe mgbochi.

Ngwongwo elu slide dị mkpa karịsịa n'ihi na oyi akwa agarose ga-anọgide na-adịchaghị mma n'oge nyocha ahụ. Usoro na-akọwa mkpuchi agarose dị ka nzọụkwụ dị oke egwu, otu nyocha na-ekwu na oyi akwa agarose dị larịị, nke ọma, dị mkpa maka ịse foto na maka izere ihe ndị na-elekwasị anya na-akpata site na elu gel na-enweghị isi.

Atụmatụ igodo

Maka ihe nkiri comet assay ka ọ rụọ ọrụ nke ọma, ọtụtụ ihe imewe dị mkpa:

• 1. Ike agarose adhesion
• Ihe mmịfe ahụ kwesịrị ịkwado mkpuchi agarose kwụsiri ike ka gel wee nọgide na-emebi emebi n'oge lysis, ịsacha, na electrophoresis. Protocol na-eji pre- mkpuchi na agarose emeziwanye mgbakwunye.
• 2. Ịdị mma elu na-agbanwe agbanwe
• Ọbụna elu na-enyere aka ibelata ihe osise eserese ma na-akwado ọdịdị ọdịdị comet otu n'ofe slide ahụ. Nke a dị mkpa n'ihi na usoro akara na-adabere na isi doro anya-na-nkewa ọdụ na ngosi cell na-agbanwe agbanwe.
• 3. Ndakọrịta usoro ọrụ
• Ọtụtụ ụlọ nyocha na-ahọrọ akwachie ma ọ bụ dị njikere-iji-iji usoro slide n'ihi na ha na-ebelata oge nkwadebe ma na-enyere aka ịhazi rịzọlt n'ofe ndị ọrụ. Nke a bara uru karịsịa na ntọala dị elu -
• 4. Nkwado maka akara ngosi enwere ike imepụtaghachi
• Ntuziaka OECD na-atụ aro ka ị nweta akara akara naanị akọwapụtara nke ọma, sel a na-ahụ anya yana nyocha mkpụrụ ndụ zuru oke n'otu nlele site na iji nyocha onyonyo ma ọ bụ ọkara - sistemu akpaaka. Ọkpụkpọ slide kwụsiri ike na-enyere aka hụ na mpaghara ndị ahụ dabara maka akara.

Ụdị Usoro ọrụ

Ọkọlọtọ comet assay workflow na-amalite site na mkpuchi slide, na-esote sel na agarose na slide ahụ. Mgbe ahụ, a na-agbanye ihe nlele ahụ iji wepụ akpụkpọ ahụ na protein ndị na-edozi ahụ, mgbe nke ahụ gasịrị, DNA adịghị emerụ ahụ ma tinye ya na electrophoresis. N'ikpeazụ, DNA nwere ntụpọ ma na-enyocha ya site na microscopy fluorescence ma ọ bụ ngwanrọ nyocha onyonyo.

N'ụdị alkaline comet assay, nyocha ahụ bara uru karịsịa maka ịchọpụta nkwụsịtụ DNA strand na alkali - saịtị labile. Ntuziaka OECD na-akọwakwa akara akara site na paramita dị ka% DNA ọdụ, ogologo ọdụ na oge ọdụ, yana% DNA ọdụ a na-atụkarị aro maka nkọwa.

Ngwa

• 1. Genotoxicity Testing: The comet assay bụ ule bụ isi na batrị nke nyocha nke a na-eji nyochaa ike genotoxic nke ọgwụ ọhụrụ, kemịkalụ ụlọ ọrụ mmepụta ihe, pesticides, na ihe mgbakwunye nri. Ọ bara uru karịsịa maka ịchọpụta mmebi DNA na vivo, na-emeju ule in vitro dị ka ule Ames na chromosome aberration test.
• 2. DNA mebiri na Nchọpụta Ndozi: Nnwale ahụ bụ ngwá ọrụ dị mkpa maka ịmụ usoro nke mmebi DNA na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe dị iche iche genotoxic, gụnyere ionizing radiation, ultraviolet light, stress oxidative, and chemical mutagens.
• 3. Environmental Biomonitoring: A na-eji nyocha comet iji chọpụta mmetụta mmetọ gburugburu ebe obibi na-emetụta ndị mmadụ na anụ ọhịa. E tinyela ya n'ụdị dị iche iche nke sentinel, gụnyere azụ, nnụnụ, na invertebrates, iji nyochaa mmetọ gburugburu ebe obibi na gburugburu mmiri na terrestrial.
• 4. Nchọpụta Ọrịa Cancer: A na-eji nyocha ahụ amụ ọrụ nke mmebi DNA na enweghị ike nke genomic na mmepe cancer na ọganihu. A na-ejikwa ya nyochaa ịdị irè nke ọgwụgwọ ọrịa cancer nke na-arụ ọrụ site n'imebi DNA mebiri emebi na mkpụrụ ndụ tumo.
• 5. Nnyocha ịka nká: Ịchịkọta mmebi DNA bụ ihe e ji mara ịka nká. A na-eji nyocha comet iji nyochaa mmekọrịta dị n'etiti mmebi DNA, ịka nká, na afọ-ọrịa ndị metụtara.

Mmechi

Ihe slide comet assay bụ akụkụ bụ isi nke nyocha mmebi DNA nke a pụrụ ịdabere na ya na nke enwere ike imepụtagharị. Ogo elu ya, ndakọrịta agarose, na njikwa nkwụsi ike niile nwere mmetụta dị ukwuu na arụmọrụ nyocha. Maka ụlọ nyocha ndị chọrọ nsonaazụ nyocha comet na-agbanwe agbanwe, nke a na-ebipụta, ịhọrọ nke ọma - ahaziri, dị elu - usoro slide precoated dị mma bụ itinye ego dị oke mkpa nke nwere ike melite arụmọrụ arụmọrụ, belata mgbanwe nnwale, ma kwado nkwubi okwu sayensị ziri ezi na ntụkwasị obi. Ka ngalaba nke mkpụrụ ndụ ihe nketa toxicology na-aga n'ihu, teknụzụ comet assay slide teknụzụ ga-aga n'ihu na-aga n'ihu, yana mmepe na-aga n'ihu na-elekwasị anya n'ịkwalite mmepụta, nhazi ọkwa, yana ndakọrịta na sistemụ onyonyo na nyocha akpaghị aka.

FAQ

Q1: Gịnị bụ ihe nkiri comet assay nke ejiri mee ihe?
A na-eji slide assay comet na-etinye sel ma ọ bụ nuclei na agarose ma mee ka nlele ahụ kwụsie ike n'oge lysis, electrophoresis, ntụpọ, na onyonyo. Nke a na-enye ohere ka a na-ahụ anya na nyocha nke nbibi strand DNA n'ọkwa otu-sel.

Q2: Gịnị kpatara mkpuchi slide ji dị mkpa na nyocha comet?
Mkpuchi ihe mmịfe na-eme ka adhesion agarose dịkwuo mma ma na-enyere aka idowe akwa gel dị larịị ma kwụsie ike. Nke a na-akwado onyonyo ka mma ma belata nsogbu njikwa n'oge usoro nyocha.

Q3: A na-ejikarị slides comet assay precoated?
Ee. Protocol e bipụtara na-akọwa ihe mmịfe microscope nke nwere agarose, yana ihe mmịfe emere nke emebere azụmaahịa dịkwa maka ojiji na usoro ọrụ comet assay.