Ina whakararu te kikorangi metilīni i te whakamātautau e whakamahia ana hei ine i a ia

Ka whakatipu tētahi taiwhanga i ngā kukune ika hepara ki roto i te kikorangi metilīni i te kukūtanga tiaki kararehe paerewa, kātahi ka ine i te pākia mā ngā ara e rua. I te rā tuatahi, ka pūrongo tētahi pūtātari Seahorse kei te piki te whakapau hāora. I ngā rā tuawhā me te tuarima, ka pūrongo tētahi pereti Alamar Blue kei te heke te whakapau pūngao. Ko taua waikano tonu, ko taua kāwai ika tonu, engari he tauaro ngā whakatau. Kātahi ka puta te whakamātautau whakahaere hūmārie: he pōuri ake hoki te pānuitanga o ngā puna e mau ana i te kikorangi metilīni me te tauhohe Alamar Blue, kāore rawa he ika, i tō te tauhohe anake. Nā te koiora tētahi wāhanga o te hua. Nā te waikano e māka ana i tōna ake whakamātautau tētahi atu wāhanga. Ko taua wehenga, i tuhia ki tētahi rangahau Communications Biology o 2025 mō te kikorangi metilīni i ngā ika hepara e whanake ana, te āhua noa o te whakararu inenga a te kikorangi metilīni. Koia hoki te take me whai whakamātautau whakahaere ngā waikano hohe hāoratanga-whakaitinga, kāore e hiahiatia ana mō ngā pūhui whakamātautau noa.

He aha tonu tā te inenga resazurin e pūrongo ai

He ingoa waitohu a Alamar Blue mō tētahi wairewa resazurin, nō reira he whakaahua ngā kupu whakararu Alamar Blue a te kikorangi metilīni me te whakararu inenga resazurin a te kikorangi metilīni i taua tukinga kotahi. He kikorangi te resazurin kua hāoratia, ā, he iti rawa tōna whārohirohi. Ka whakaiti ngā pūtau hohe ā-pākia i a ia kia riro hei resorufin, he māwhero, he kaha hoki te whārohirohi i te takiwā o te whakahihiko 560 nm me te tukunga 590 nm. Ko ngā awhe e taea ana te whakamahi ko te whakahihiko 530 ki te 570 nm me te tukunga 580 ki te 610 nm, e ai ki te kawa pereti-iti a te kaiwhakanao. Ki te kaha ake te whakaitinga, ka huri te resorufin hei dihydroresorufin, he ngoikore tōna whārohirohi. Nō reira, ki te nui rawa ngā pūtau i te puna, ki te roa rawa rānei te wā, ka piko whakararo te ānau whakatikatika i tōna pito runga.

Ko te nuinga o ngā irahiko ka ahu mai i te NADH me te NADPH mā ngā whākōkī reductase me te diaphorase o te pūtau, kei te wai pūtau, ngā microsome, me ngā pungao. Nā reira, ko tā te pānuitanga he whakaatu i te āheinga whakaiti kua whakakotahitia, ehara i te tatau i ngā pūtau ora, ehara hoki i te inenga pungao. Ka tohu pea te tohu teitei ake i te maha ake o ngā pūtau, i te nui ake o te rere whakaiti mō ia pūtau, i ēnei mea e rua rānei. Ka whakarerekē tētahi pūhui e whakahuri ana i te ara o te NADH i te tohu me te kore e whakamate i tētahi pūtau kotahi. Puritia taua kōrero ki te taha, nā te mea he mahi matua mā te kikorangi metilīni te whakahuri i te ara o te NADH.

E rua ngā ara whakararu, kotahi te tohu kua heke

Ka whakakotiti te kikorangi metilīni i te pānuitanga resazurin mā ngā ara motuhake e rua, ā, mā tētahi o ēnei anake ka heke te whārohirohi e kitea ana.

He ara rama te ara 1. He kaha te ngongo rama a te kikorangi metilīni i te wāhanga whero, me tōna pae monomer i te takiwā o te 660 ki te 665 nm, me tētahi pakihiwi dimer i te takiwā o te 607 ki te 610 nm. Ka toro whakamuri te hiku o taua pakihiwi ki te tukunga resorufin i te takiwā o te 590 nm. Ka ngongo te waikano i te ara rama i tētahi wāhanga o te rama whakahihiko, ā, ka ngongo anō i tētahi wāhanga o te whārohirohi kua tukuna. He pānga tātari-roto tēnei e whakapōuri ana i te puna ahakoa kāore e rerekē te hanga resorufin. Ka neke hoki te tūāwhiorangi i runga i te āhua o te waikano: ka wehe tētahi tātari tauwehe o ngā tūāwhiorangi kikorangi metilīni i roto i te wai me ngā wairewa cyclodextrin i te monomer, te dimer, me te pūhui kaiwhakamanuhiri-manuhiri hei momo matū e toru, he rerekē ō rātou whakarea ngongo mai i te 500 ki te 700 nm. Nō reira, ka rerekē te nui o te hapa rama i runga i te kukūtanga, te whakahuihui, me te here. I roto i te whakamātautau, wehewehea te whakangoikore rama noa i te tinei ā-rāpoi mā te whakamātautau whakaoranga tāpiritanga e whakaahuatia ana i raro nei, nā te mea he rerekē ngā whakatikanga.

Ko te ara 2 he whakataetae hāoratanga-whakaitinga. Ka tango te kikorangi metilīni kua hāoratia i ngā irahiko mai i te NAD(P)H hei hanga i te kikorangi metilīni leuco kore tae. Ka hāora anō te hāora ā-rāpoi i tēnei kia hoki ki te āhua kikorangi, me te hanga hua hāora kua whakaitia, tae atu ki te hauwai peroxide. Ka whakapau ia takahanga o taua huringa i ngā wae ōrite whakaiti ka whakaiti kē i te resazurin mēnā kāore taua huringa, ā, ka whakapau hoki i te hāora me te hanga peroxide i te taha. Nō reira, ka tohu pea te tohu Alamar Blue kua heke i te haere o ngā irahiko mā te huringa kikorangi metilīni, kaua ki roto i te resorufin. Ka pānuitia tēnei hei pākia kua heke i runga i te pereti, ahakoa he aha ngā mahi a ngā pūtau. Ka whakamārama taua matū anō i tētahi mahanga tauaro i ngā taputapu hāora: ka akiaki te waikano kua whakaitia, ina hāoratia anō e te hāora, i te whakapau hāora ā-matū ahakoa kāore he koiora. Nō reira, me whai tauira waikano pātea hoki ngā inenga hā, pēnei i tā te whakataurite o ngā pānuitanga hāora, ATP, me te pūmanawa kiriuhi e whakatakoto ana mō ngā pungao.

Ka pono tahi pea te tohu kua rerekē me te koiora kua rerekē

He tino whai tikanga te rangahau ika hepara nā te mea kāore ia e whakaae ki te kōwhiringa māmā o tētahi, o tētahi atu rānei. He tūturu te koiora: i piki te whakapau hāora taketake i ngā kukune 1-rā te pakeke i ngā kukūtanga tiaki e rua; i piki te hā e hono ana ki te ATP i te kukūtanga raro; i piki te pūmanawa kiriuhi i inea mā te pūoko JC-10 i te rā tuarua; ā, i heke ngā tuhinga RNA o ngā huinga pungao i ngā rā tuatahi me te tuawhā. He tūturu hoki te hapa inenga: ahakoa i horoia ngā ika kia toru ngā wā i mua i te takahanga Alamar Blue, i kitea tonu he poke kikorangi i te upoko, te waha, me te rohe hongi. I ngaro hoki i ngā puna kore pūtau me te waikano me te tauhohe tētahi rahinga iti, engari hira, o te whārohirohi i a rātou anake. He nui ake ngā hekenga Alamar Blue o te rauropi i ngā rā tuawhā me te tuarima i te pānga kore pūtau, nō reira i whakamāori ngā kaituhi i te hua hei panonitanga pākia tūturu e tirohia ana mā tētahi arotahi paku kōtiti. Koinei te tauira whakaaro tika. Ko tētahi matū huri hāoratanga-whakaitinga e ngongo ana hoki i te rama hei waikano kikorangi, ka whakararu i te ara e rangahaua ana me te matū o te pūrongo i te wā kotahi. Ko tā ngā whakamātautau whakahaere he ine i te wāhanga nā ia pānga, kaua e kī he mea pohewa tētahi o aua pānga. He akoranga e pā ana ka puta i te rangahau here cyclodextrin: i te wā i mau ai te rāpoi kaiwhakamanuhiri i te waikano, i memeha tahi te poke me te whakahohe i te tango kūhuka. He taunakitanga tēnei i hiahia te pānga pākia ki te pāhekoheko hāngai ki te waikano, kaua ki te ngangau hāoratanga-whakaitinga whānui. Ko te whakataurite i tētahi pānuitanga ā-ahupūngao o te waikano ki tētahi pānuitanga mahi te mahi e wehe ai te pūtake i te hapa inenga, ā, ka whakawhanake anō te aratohu pānui kerēme pākia e whakamahi ana i te waikano i taua tikanga.

Ngā whakamātautau whakahaere e wehe ai i ēnei

Whakahaerehia ēnei i mua i te whakamāori i tētahi hua kikorangi metilīni mai i tētahi pereti resazurin. Me tino ōrite ngā kukūtanga, te taiao whakatipu, te wā whakapao, me te whakamārama rama ki te whakamātautau pūtau, nā te mea e whakawhirinaki ana ia ara i runga ake nei ki ēnei.

Ko ngā whakamātautau whakahaere 3 me te 4 te takirua ka mahue i te nuinga o ngā tuhinga, ā, e hiahiatia ana e te nuinga o ngā tuhinga kikorangi metilīni. Ka hopu te whakamātautau whakahaere 3 i te matū, ka hopu te whakamātautau whakahaere 4 i ngā pānga rama. Mā rāua tahi e whakatau ngā paenga o te hapa inenga i mua i tētahi kerēme koiora.

Ngā pānuitanga motuhake, me ngā here ake o tēnā, o tēnā

Kāore tētahi inenga tuarua kotahi e whakamana i te mea tuatahi. Ka karo ia pānuitanga motuhake i tētahi ara, engari ka noho whakaraerae tonu ki tētahi atu. Nō reira, kōwhiria te huinga kāore e īnaki ana ōna wāhi kāpō. Ka whakamārama te tūtohi kōwhiri inenga mō ngā pūhui hohe hāoratanga-whakaitinga i te arorau takirua, ā, ka āta whakamārama te aratohu pūoko pūmanawa kiriuhi i ngā tūpato mō te whārohirohi.

Ka whai te inenga whakapau hāora Seahorse i ngā irahiko ki te hāora, kaua ki roto i te resorufin. Ka karo tēnei i te matū pūrongo katoa, engari ka whakapau hoki te waikano i te hāora mā te matū. Nō reira, me whai tauira waikano pātea kāore he pungao, kei roto i te taiao whakatipu oti. Ka whakamahi ngā inenga luciferase ATP i te tīrama koiora, kaua i te whārohirohi resorufin, nā reira ka karo i te īnakitanga rama me te whakataetae reductase. Ko te utu mō tēnei, he pūrongo i te taurite o te puna ATP, kaua i te rere hanganga ATP. Ka whakautu ngā pūoko pūmanawa kiriuhi pēnei i te TMRM me te JC-10 i te pātai mēnā e mau tonu ana te rōnaki utanga hiko. Heoi, ka whārohirohi rātou i taua awhe rama kitea e ngongo ai te waikano, nō reira ka whakapōraruraru hoki te poke kikorangi toenga i a rātou, ā, me whai tauira waikano-anake pātea i ia rahinga waikano. Ka karo rawa ngā inenga tuhinga RNA o ngā wāhanga iti o ngā huinga hā i ngā pānga rama, ā, ka whakakotahi i ngā pānga puta noa i ngā hāora. Ka tautoko tēnei i ngā pānuitanga pereti e whāia ana i te tauine meneti.

Nō reira, ko te huinga iti rawa e pono ai tētahi kerēme pākia kikorangi metilīni: he hua resazurin me ngā whakamātautau whakahaere 1 ki te 4, me tētahi ine mutunga ehara i te resazurin e ahu mai ana i tētahi mātāpono ahupūngao rerekē, me tētahi kōrero mārama mēnā e whakaata ana te panonitanga i te maha o ngā pūtau, te rere whakaiti mō ia pūtau, ēnei mea e rua rānei. Ki te iti iho i tēnā, kāore te kaipānui e āhei ki te wehe i te pākia ngoikore ake i te puna pōuri ake.

Pepamahi whakahaere whakararu

Tāruatia tēnei pepamahi ki te pukapuka taiwhanga mō ia whakamātautau e whakakotahi ana i te waikano me tētahi pūrongo hāoratanga-whakaitinga. Koinei te rauemi taketake mō tēnei tuhinga: whakakīa tētahi rārangi mō ia kukūtanga waikano.

A. Ngā ara tohu. Mō ia kukūtanga, tuhia: te āhua monomer, dimer rānei e tūmanakohia ana (he iti ngā mikoromōra, he tekau tini ngā mikoromōra me runga ake rānei), te ngongo rama o ngā ihirangi whakamutunga o te puna i te 590 nm i roto i tētahi puna kore pūtau, me te ōrau whakaoranga o te tāpiritanga resorufin. Ki te heke te whakaoranga ki raro i te āhua 90 ōrau, whakaarohia ngā uara whārohirohi katoa i taua rahinga hei uara kua whakaitia nā te rama, kaua hei uara koiora tino tika.

B. Tirohanga matū. Tuhia te whārohirohi kore pūtau o te waikano me te tauhohe hei ōrau o te pae taketake tauhohe-anake i ia wā pānui. Ki te tipu te rerekētanga i roto i te wā, kei te tauhohe te waikano ki te pūnaha pūrongo i te wā o te inenga, ā, ka whakapohehe ngā pānuitanga mutunga-anake. Whakaarohia ngā pānuitanga e whai ana i te huringa i roto i te wā, tētahi pūrongo kē rānei.

C. Tirohanga whakawhiti koiora. Whakaingoatia te pānuitanga motuhake kua kōwhiria mō tēnei whakamātautau, tōna ake tauira waikano pātea, me te hua e matapaetia ana mēnā he tūturu te hua resazurin. Mā tētahi hekenga pākia tūturu e neke tētahi ine mutunga motuhake ki tētahi ahunga ōrite. Ki te whakahē te ine mutunga motuhake, kaua anō e whakawhirinaki ki tētahi o ngā hua; hoahoatia anō te whakamātautau, kaua e tango i te toharite o ngā mea e rua.

D. Rerenga whakatau. Tuhia tētahi rerenga kōrero e pēnei ana: i te rahinga me te wāhanga kua tohua, i rerekē te tohu resazurin mā te rahinga kua tohua, ā, he hapa inenga kore pūtau te rahinga kua tohua o taua panonitanga, me te ine mutunga motuhake e (whakaae ana, whakahē ana rānei). Ki te pātea tētahi wāhi, kāore anō kia oti te whakamātautau.

He pūoko pākia whai hua tonu te kikorangi metilīni, ā, ka whakaatu te rangahau ika hepara i ngā pānga pungao tūturu i ngā rahinga e ringihia ana ki te waikeri ia rā e ngā whare maha. Ko tā te waikano noa he tono kia inea ia mā ngā taputapu kāore e taea e ia te whakararu. Hoatu he puna waikano-anake, he whakaoranga tāpiritanga, me tētahi pānuitanga e hangaia ana ki runga i te ahupūngao rerekē, ā, ka mutu te whakararu hei mahanga, ka tīmata hei rahinga kua āpitihia he tau.