Buku Logwiritsa Ntchito la IZON qEV2 Extra Cellular Vesicles

IZON qEV2 Zowonjezera Ma Cellular Vesicles

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Zizindikiro zonse za chipani chachitatu ndi katundu wa eni ake.

Izon Science Ltd PO Box 9292 Addington Christchurch 8024 New Zealand
Telefoni: + 64 3 357 4270
Email: support@izon.com
Website: www.izon.com

MISONKHANO YOTANTHAUZIRA NDI KULEMBA

Onetsetsani kuti mukutsatira mfundo zodzitetezera zomwe zaperekedwa mu bukhuli. Chitetezo ndi zidziwitso zina zapadera zidzawonekera m'mabokosi ndikuphatikiza zizindikiro zomwe zafotokozedwa mu Gulu 1.
Gulu 1: Zizindikiro Zachitetezo ndi Zowopsa

Chizindikirochi chikuwonetsa upangiri wanthawi zonse wamomwe mungasinthire njira kapena kupangira njira zomwe mungatsatire pazochitika zinazake.
Chizindikirochi chimasonyeza kumene tiyenera kusamala kwambiri.

Gulu 2: Mawu Ogwiritsidwa Ntchito M'bukuli

TERM	CHIFUKWA
Buffer voliyumu (Volumu yopanda kanthu)	Voliyumu yamadzimadzi yomwe imagwirizana ndi voliyumu isanachitike Pulumu Yotolera Yoyeretsedwa (PCV); bukuli likusonkhanitsidwa zodziwikiratu pachitsime chapakati. Voliyumu ya buffer ndi yosiyana pamtundu uliwonse.
Chromatography	Njira yomwe imagwiritsidwa ntchito makamaka pakulekanitsa zigawo za asample. Zigawozo zimagawidwa pakati pa magawo awiri; imodzi imakhala yoyima pomwe inayo imakhala yoyenda. Gawo loyima limakhala lolimba, lamadzimadzi lokhazikika, kapena gel. Gawo loyima likhoza kudzazidwa mumzere, kufalikira ngati wosanjikiza kapena kugawidwa ngati filimu. The mafoni gawo akhoza kukhala mpweya kapena madzi.
Voliyumu yazanja	Voliyumu yophatikizika yazinthu zopakidwa ndi voliyumu ya buffer (imatha kutchedwa voliyumu ya bedi).
Degassing	Kuchotsa mpweya kumaphatikizapo kuchotseratu mpweya wothira muvuni kuti “wiritsani” kuti muchotse mpweya wochuluka wosungunuka monga kugwiritsa ntchito vacuum mu botolo.
Kuthamanga kwa mlingo	Volumetric otaya mu mL/mphindi wa madzi chonyamulira.
Purified Collection Volume (PCV)	Voliyumu yomwe imalowa m'malo mwa voliyumu ya bafa, yokhala ndi tinthu tating'ono ta chidwi toyeretsedwa kuchokera ku s zodzazaample. PCV ndi yosiyana pamtundu uliwonse.
Kubwezeretsa	Ntchito ya percentage ya ma vesicles omwe amatuluka muzanja poyerekeza ndi zomwe zidalowa.
Smart Column	Mzere wa qEV wokhala ndi chipangizo chophatikizika cha RFID. Chip ichi chimagwiritsidwa ntchito ndi chida cha AFC kuzindikira mtundu wa mzati womwe umayikidwa pachidacho. Chip ichi sichimakhudza kugwiritsa ntchito pamanja pagawo la qEV.

CHITETEZO NDI ZOOPSA

Onani ku Satifiketi Yachitetezo kuti mugawidwe ndikuyika zilembo zangozi ndi zidziwitso zofananira ndi chitetezo. Tsamba la Chitetezo cha Magawo a qEV SMART lili pa
izon.com/sds

Ngozi

qEV SMART columns ndi mankhwala a labotale. Komabe, ngati biohazardous sampZomwe zilipo, tsatirani Makhalidwe Abwino a Laboratory (cGLP) ndipo tsatirani malangizo amdera lanu okhudzana ndi labotale yanu ndi komwe muli.

Kutaya kwa Biohazardous Material
Gawo la qEV SMART lili ndi <0.1% sodium azide, yomwe imatha kupha munthu ikamezedwa kapena kukhudzana ndi khungu. Chonde review malangizo ndi chenjezo zotsatirazi musanagwiritse ntchito gawo lililonse la qEV SMART:

kupewa:

1. Osalowa m’maso, pakhungu, kapena pa chovala;
2. Sambani khungu bwinobwino mukatha kulisamalira.
3. Musadye, kumwa, kapena kusuta pamene mukugwiritsa ntchito mankhwalawa.
4. Pewani kutulutsa mankhwala ku chilengedwe.
5. Valani magolovesi oteteza ndi zovala; tsatirani njira zodzitetezera ku labotale.

Poyankha

1. NGAMAKUMWA: imbani nthawi yomweyo ku POISON CONTROL CENTRE/Doctor.
2. NGATI PAKHUMBA: Sambani pang'onopang'ono ndi sopo ndi madzi ambiri ndipo nthawi yomweyo imbani POISON CONTROL CENTRE/Doctor.
3. Chotsani nthawi yomweyo chovala chilichonse chomwe chawonongeka ndikuchapa musanagwiritsenso ntchito.
4. Sonkhanitsani kutaya kulikonse ndikutaya moyenera.

Kuti mumve zambiri, onani Zolemba za MSDS za magawo a Izon qEV SMART: izon.com/sds

Chenjezo
Sodium azide imatha kupha munthu ikamezedwa kapena kukhudzana ndi khungu. Zitha kuwononga ziwalo zaubongo kudzera mukuwonekera kwa nthawi yayitali kapena mobwerezabwereza. Ndiwowopsa kwambiri ku moyo wam'madzi wokhala ndi zotsatira zokhalitsa.

Onetsetsani kuti mukutsatira malangizo otsatirawa ndikutsatira malangizo apafupi a labotale yanu ndi malo okhudzana ndi kugwiritsidwa ntchito ndi kutaya.

Zosamalitsa Zonse:

• Nthawi zonse muzivala magolovesi a labotale, malaya, ndi magalasi oteteza chitetezo okhala ndi zishango zam'mbali kapena magalasi.
• Manja anu akhale kutali ndi pakamwa panu, mphuno, ndi maso.
• Tetezani kotheratu kudula kapena zilonda zilizonse musanagwiritse ntchito ndi zinthu zomwe zitha kupatsirana kapena zowopsa.
• Sambani m'manja mwanu bwinobwino ndi sopo mutagwira ntchito ndi chinthu chilichonse chomwe chingapatsire matenda kapena choopsa musanachoke mu labotale.
• Chotsani mawotchi ndi zodzikongoletsera musanagwire ntchito pa benchi.
• Kugwiritsa ntchito magalasi olumikizana sikuvomerezeka chifukwa cha zovuta zomwe zingabwere panthawi yosamba maso mwadzidzidzi.
• Musanachoke mu labotale, chotsani zovala zodzitetezera.
• Osagwiritsa ntchito golovu polemba, kuyankha lamya, kuyatsa cholumikizira magetsi, kapena kumenya anthu opanda magolovesi.
• Sinthani magolovesi pafupipafupi.
• Chotsani magolovesi nthawi yomweyo pamene ali ndi kachilombo.
• Osawonetsa zinthu zomwe sizingawonongedwe moyenera kuzinthu zomwe zitha kupatsirana kapena zowopsa.
• Mukamaliza ntchito yokhudzana ndi zinthu zomwe zitha kupatsirana kapena zowopsa, chotsani malo ogwirira ntchito ndi mankhwala ophera tizilombo kapena njira yoyeretsera (1:10 dilution of bleach wapakhomo akulimbikitsidwa).

Tayani zinthu zotsatirazi zomwe zitha kukhala ndi kachilombo molingana ndi malamulo a labotale amderalo, madera, ndi dziko:

• Biological Samples
• Zowonjezera
• Zotengera zomwe zimagwiritsidwa ntchito kapena zinthu zina zomwe zitha kuipitsidwa

yosungirako
Kusintha kwachangu kwa kutentha kuyenera kupewedwa, chifukwa izi zitha kuyambitsa thovu mu bedi la gel.
Mizati ya qEV yosagwiritsidwa ntchito imatha kusungidwa kutentha. Mizati ya qEV yogwiritsidwa ntchito ikhoza kusungidwa kutentha kwa firiji popereka kuti yatsukidwa molingana ndi malangizo omwe ali pachikalatachi ndikusungidwa mu 20% ethanol kapena 0.05% w/v sodium azide. Ngati njira zoyenera sizikupezeka kuti zisungidwe kutentha kwa chipinda, ndiye kuti mizati imatha kusungidwa pa +4 mpaka +8 ° C ikagwiritsidwa ntchito.

Kutaya
Zosungiramo zinyalala ziyenera kutayidwa motetezeka. Kuchuluka kwa sodium azide pakapita nthawi mu mapaipi amkuwa kungayambitse kuphulika.

MAU OYAMBIRA KUKUKULU KUKHALA KOMA CHROMATOGRAPHY

paview
Zigawo za Izon's qEV SMART Size exclusion chromatography (SEC) zimalekanitsa tinthu ting'onoting'ono potengera kukula kwake pamene akudutsa pagawo lodzaza ndi porous, polysaccharide resin. Pamene mamolekyu amalowa mu utomoni, tinthu tating'onoting'ono timatsekeredwa mu pores ndipo kutuluka kwawo kuchokera pamzati kumachedwa (Mkuyu 1C). Pamene madzi akutuluka mzati, zigawo zotsatizana zimasonkhanitsidwa. Tinthu tating'ono ting'onoting'ono tomwe timagawira tizigawo ting'onoting'ono totengera kukula kwake, ndi tinthu tating'onoting'ono tomwe timatulutsa tinthu tating'onoting'ono tomwe timatuluka m'gawoli.
Mzere wodzaza ndi wofanana ndi buffer, yomwe imadzaza ndime. Voliyumu yonseyi imakhala ndi utomoni wokhazikika (gawo lokhazikika) ndi buffer yamadzimadzi (gawo loyenda). Popeza tinthu tating'onoting'ono sitimangiriza ku utomoni, mawonekedwe a buffer sangakhudze kwambiri chigamulo (kuchuluka kwa kulekana pakati pa nsonga).

Chithunzi 1: Njira ya SEC (A) Chithunzi chojambula cha mkanda wa resin wokhala ndi kukula kwa electron microscopic. (B) Chojambula cha sample particles diffusing mu pores wa tinthu. (C) Kufotokozera mojambula za kulekana: (i) sample imayikidwa pakhoma; (ii) kachigawo kakang'ono kwambiri (chikasu) kachedwa kwambiri kuposa kachigawo kakang'ono (kofiira); (iii) gawo lalikulu kwambiri limachotsedwa koyamba pamzati. Kukula kwa bandi kumapangitsa kuchepetsedwa kwakukulu kwa magawo a tinthu panthawi ya chromatography. (D) Chromatogram yamakono. Kuchokera ku: GE Healthcare ndi Biosciences. (ndi). Kupatula Kukula kwa Chromatography Mfundo ndi Njira [Kabuku]. Uppsala, Sweden. Adafikira mu June 2019.

Ntchito Yogwiritsidwa Ntchito

Mizati ya Izon qEV SMART imapatula ma vesicles owonjezera kuchokera ku biological samples. Magawo a qEV SMART ali ndi tchipisi ta RFID kuti agwiritse ntchito ndi Izon Automatic Fraction Collector (AFC). Ma chips awa adzatero
osakhudza kugwiritsa ntchito pamanja. Mzerewu uyenera kugwiritsidwa ntchito m'ma laboratories ofufuza ndi akatswiri kuti agwiritse ntchito kafukufuku wokha. Gawo la qEV silinapangidwe kuti liziwunikira matenda ndipo siliyenera kugwiritsidwa ntchito popanga chisankho chamankhwala.
Magawo a qEV SMART adapangidwa kuti azipatula ndi kuyeretsa ma vesicles ku ma biological s ambiriampLes, kuphatikizapo:

• Seramu
• Plasma
• Malovu
• Mtsinje
• Cerebrospinal Fluid (CSF)
• Ma cell media media

ZINDIKIRANI: kwambiri 'yaiwisi' samples sizingayendetsedwe mwachindunji pamizere ya qEV SMART ndikuwunikidwa ndi TRPS popanda kukonzekera kwina monga centrifugation ndi masitepe okhazikika. Lumikizanani ndi Izon Support Center kuti mupeze malingaliro ndi ma protocol.

Kuyerekeza kwa qEV/35nm ndi qEV/70nm mndandanda
Magawo onse a qEV SMART akupezeka mumodzi mwa magawo awiri odzipatula, mndandanda wa qEV/35nm ndi qEV/70nm. Mndandanda wa mizati ya qEV/35nm nthawi zambiri umagwira ntchito bwino ngati tinthu tating'onoting'ono tokhala patali ndi 110 nm m'mimba mwake, pomwe mndandanda wa qEV/70nm wa mizati nthawi zambiri umachita bwino pamene tinthu tating'onoting'ono tokhala patokha ndi lalikulu kuposa 110 nm m'mimba mwake. (onani Gulu 3). Pakuti mulingo woyenera kuchira particles pakati 35 ndi 350 nm ndi qEV/35nm mndandanda ndime tikulimbikitsidwa. Kuti muchepetse kuchira kwa tinthu ting'onoting'ono pakati pa 70 ndi 1000 nm, gawo la qEV/70nm limalimbikitsidwa.

Chithunzi 2. Kubwezeretsa kwapang'onopang'ono ndi kukula kwa qEV/35 mndandanda wa mndandanda poyerekeza ndi mzere wa qEV/70 (mzere wapinki).

Table 3: Zofotokozera za qEV/35nm ndi qEV/70nm Series

	qEV/35nm SERIES	qEV/70nm SERIES
Target Particle Kukula	35 nm mpaka 350 nm	70 nm mpaka 1000 nm
Mulingo Wabwino Wobwezeretsa	Tinthu <110 nm	Tinthu > 110 nm

Zithunzi za qEV2

Gulu 4: Zofotokozera za qEV2

Dzinalo	qEV2
Zotsatizana	qEV2/35nm	qEV2/70nm
Kukula koyenera kolekanitsa	35-350 nm	70-1000 nm
Nthawi Yothamanga	~ Mphindi 30
Voliyumu yazanja	45.1 mL
Sampndi load volume	Mpaka 2 ml *
Mulingo woyenera wagawo	2 mL
Voliyumu ya buffer **	14.1 mL
Voliyumu yamphamvu	60 mL
Voliyumu yosonkhanitsidwa yoyeretsedwa **	8 mL
Chiwopsezo cha EV pambuyo pa voliyumu ya buffer **	4 ± 2 mL
Kutentha kogwira ntchito	18 ku 24 ° C
gawo lotetezedwa	PBS
Kukula kwakukulu kodutsa	1 μm
Kukula kwa zosefera zapamwamba ndi pansi	20 μm
pH yokhazikika yogwira ntchito	3 - 13
pH kukhazikika kuyeretsa-pamalo (CIP)	2 - 14
Nthawi ya alumali (ngati yasungidwa bwino)	miyezi 12

*Izon amalimbikitsa kutsitsa 2mL kuti muwonjezere chiyero.
**Makhalidwe a plasma yamunthu samples yekha.

qEV2 Magwiridwe Antchito

Monga momwe chithunzi 3 ndi 4 pansipa, particles zosakwana 70 nm zambiri elute mochedwa kuposa PCV pa qEV/70nm, pamene particles zazikulu kuposa 35 nm ndi anagwidwa mu zone pa ndime qEV/35nm. A apamwamba achire mu PCV wa particles zazikulu kuposa 70 nm amapezeka pa qEV/70nm mndandanda mizati poyerekeza ndi qEV/35nm mndandanda (mkuyu. 3). Mapuloteni nthawi zambiri amatuluka pang'onopang'ono pamndandanda wa qEV/35nm. Mapuloteni apamwamba mu PCV makamaka chifukwa cha kuwonjezeka kwa mapuloteni omangidwa ndi EV. Chithunzi 3: Kuyerekeza kwa plasma protein elution and recovery levels of 69 nm liposomes pakati pa qEV2/35nm ndi qEV2/70nm.Chithunzi 4: Kuyerekeza kwa plasma protein elution and recovery levels of 200 nm particles pakati pa qEV2/35nm ndi qEV2/70nm.

qEV2 EV Elution Profile

Kutulutsa kwa ma vesicles pachimake pa 6 mL ± 2 mL pambuyo pa voliyumu ya buffer, kwa 2 mL s.ample voliyumu ndikutolera tizigawo ta 2 ml. Chithunzi 5 chikuwonetsa kutuluka kwa ma vesicles pamene 2 mL ya plasma sample imakwezedwa pagawo la qEV2/35nm.
Ma EV ambiri nthawi zambiri amakhala mu 8 ml. Ngati chiyero chapamwamba chikufunika, sonkhanitsani 6 ml yokha yoyamba. Wogwiritsa ntchitoyo amasankha pakati pa kukulitsa kuchira mwa kusonkhanitsa voliyumu yayikulu kapena kukulitsa chiyero mwa kusonkhanitsa voliyumu yocheperako.
Kutulutsa kwa mapuloteni a plasma kumakhala pang'onopang'ono, kumachokera ku 10 - 38 mL pambuyo pa voliyumu ya buffer. Ma vesicles aliwonse omwe adachira kupitilira 8 mL amakhala ndi kuipitsidwa kwa protein yambiri ndipo sangakhale oyenera kuwunika kumunsi kwa mtsinje chifukwa chakuchepa kwawo.
Chizindikiro cha protein elution profiles ikhoza kupezeka poyang'anira kuyamwa pamtunda wa 280 nm. Muyeso wolondola wa kuchuluka kwa mapuloteni ukhoza kupezeka pogwiritsa ntchito colorimetric protein assay. Chithunzi 5: Wodziwika bwino wa elutionfile kwa qEV2/35nm ndime yokhala ndi 2 mL ya plasma yodzaza; Mapuloteni amatuluka mochedwa kuposa ma vesicles. Kuchuluka kwa vesicle kunayesedwa pogwiritsa ntchito TRPS ndi mapuloteni okhudzana ndi mapuloteni ndi kuyamwa pa 280 nm.

qEV2 Sample Input Volume Effects ndi

Kuchira Mitengo
Zotsatira za Sample Input Volume pa EV Elution Profile
Kutsegula apamwamba sample voliyumu imapangitsa kuti pakhale chiyero chochepa m'mavoliyumu apambuyo a vesicle, kuphatikizika kwakukulu pakati pa mapuloteni ndi EV elution nsonga, ndi nsonga yapamwamba ya mapuloteni mkati mwa PCV. Chithunzi 6 chikuwonetsa zotsatira za kukweza ma voliyumu akuwonjezeka. A sample voliyumu ya 4 mL imapangitsa kuti tinthu ting'onoting'ono timene tituluke mochedwa kuposa 8 mL PCV. Onani kuchedwa kwa EVs mu 4 mL sample.
Mulingo woyenera bwino sample voliyumu yoyera pa qEV2 ndi 2 mL, zomwe zimapangitsa kuti ma vesicles alowe mu 8 mL PCV.
Kutayika kwa ma vesicles kumachitika ndi sampKuchuluka kwa 4 mL kumawonjezera kuchuluka kwa vesicle. Ma EV omwe amasonkhanitsidwa kunja kwa 8 mL PCV savomerezedwa kuti afufuze kumtunda komwe kumayenera kukhala koyera kwambiri. Chithunzi 6: Zotsatira zakukweza ma voliyumu okulirapo zikuwonetsedwa mu elution profiles kuchokera potsegula 2 mL ndi 4 mL ya CPC200 particles (OD600) ndi plasma (OD280).

Zotsatira za Mtundu wa Resin pa Protein Elution Profile
PCV pa qEV2 ili ndi mapuloteni ochepa kwambiri, ndipo mapuloteni amawonjezeka m'mavoliyumu apambuyo, monga momwe tawonetsera pa Chithunzi 7. Mapuloteni apamwamba a qEV2 / 35nm amayamba chifukwa cha kuchira kwakukulu kwa ma EV ang'onoang'ono mu PCV (onani mkuyu. 2).

Zotsatira za Sample Input Volume pa EV Dilution ndi Recovery Rate
Mlingo wobwezeretsa wa tinthu tating'onoting'ono timatengera kukula kwa tinthu tating'ono ndi magawo a qEV SMART omwe amagwiritsidwa ntchito. Onani kuyerekeza pakati pa qEV/35nm ndi qEV/70nm mndandanda pamwambapa, Zithunzi 2-4. The dilution factor zimadalira sample kutsitsa voliyumu ndi PCV yonse yophatikizidwa. Chithunzi 7: Peresentitagndi mapuloteni cf. kuyambira kuchuluka kwa mapuloteni kuchokera pakukweza 2 mL ya plasma pa qEV2. Mipiringidzo ya pinki ndi mipiringidzo ya PCV. Mipiringidzo ya qEV2/70nm ndi yapinki yoderapo komanso yabuluu wakuda. Mipiringidzo ya qEV2/35nm ndi yowala pinki komanso yopepuka yabuluu.

MALANGIZO OGWIRITSA NTCHITO MANUKO

Gawo lotsatirali likupereka malangizo ogwiritsira ntchito pamanja magawo a qEV SMART. Kuti mugwiritse ntchito mizati ya qEV SMART yokhala ndi chida cha Automatic Fraction Collector (AFC), chonde onani Buku lathunthu la AFC User Manual pa support.izon.com

Malangizo Ogwira Ntchito

Malingaliro otsatirawa aperekedwa kuti awonetsetse kuti gawo la qEV SMART likuyenda bwino:

• Centrifuge samppang'ono musanalowetse pa column. Kuti mupewe kutsekeka kwa zosefera, tikulimbikitsidwa kusefa kapena centrifuge biological sample kuchotsa zinthu zazikulu.
  • Centrifuge sampkuchepera pa 1,500 xg kwa mphindi 10 kuchotsa ma cell ndi tinthu tambiri.
  • Pang'onopang'ono sunthani supernatant ku chubu chatsopano ndi centrifuge kachiwiri pa 10,000 xg kwa 10 min.
  • Pakupatula ma microvesicle, gwiritsani ntchito mphamvu zochepa za g pa sitepe yachiwiri ya centrifugation.
• SampLes ikhoza kukhazikitsidwa musanagwiritse ntchito pamzati kapena mutatha kudzipatula ngati pakufunika. N'zotheka kuika maganizo sampkuchepera zonse zisanachitike komanso/kapena zitatha kugwiritsa ntchito gawo la qEV SMART, komabe Izon imapereka makulidwe angapo kuti achepetse kufunikira kwa pre-analytical s.ampndi concentration. Ngati ma protocol olimbikitsira akufunika, chonde lingalirani izi:
  • Kukhazikika kwa ena sampMitundu ya le ingapangitse kuti madzi ayambe kusungunuka ndi kuphatikizika kwa mapuloteni, makamaka mkodzo samples. Kukhazikika sampLes iyenera kukhala centrifuged pa 10,000 xg kwa mphindi 10 isanakweze pagawo la qEV SMART.
  • Izon amalimbikitsa kugwiritsa ntchito zida za Merck Millipore (zosefera za Amicon® Ultra Centrifugal; C7715). Gwiritsani ntchito malinga ndi malingaliro a wopanga.
  • Kukhazikika kwa sampKuchepa pambuyo pakuyeretsedwa ndi qEV kungayambitse kutaya kwa ma EV ena pa nembanemba.
• Ndibwino kugwiritsa ntchito magawo amodzi pomwe ma vesicles amawunikidwa ngati ma nucleic acid. Kugwiritsa ntchito mzere wogwiritsa ntchito kamodzi, monga Izon qEVsingle, kumachepetsa kuthekera kwa kuipitsidwa.
• Onetsetsani kuti sample buffer yakonzedwa moyenera. Kuti musunge magwiridwe antchito a ma EV, chotchingira chotenthetsera chiyenera kukhala cha kutentha kofanana ndi sampndi buffer. SEC itha kugwiritsidwanso ntchito kusinthanitsa buffer ya asample.
  • SampKutentha kwa buffer kuyenera kukhala mkati mwa kutentha kwa 18-24 ˚C (65-75 ˚F).
  • Sampma buffer ayenera kuchotsedwa mpweya komanso kutentha kwachipinda kuti pasakhale thovu la mpweya pabedi la gel osakaniza. Kusintha kwachangu kwa kutentha, mwachitsanzoampKuchotsa mizati yodzaza m'chipinda chozizira ndikuyika malo otchingira kutentha, kumatha kuyambitsa thovu la mpweya pabedi lodzaza, zomwe zimapangitsa kuti pasakhale kulekana.
  • Gwiritsani ntchito chotchinga chokhala ndi mphamvu ya ayoni ya 0.15 M kapena kupitilira apo kuti mupewe kuyanjana kulikonse kosafunika kwa ayoni pakati pa molekyulu ya solute ndi matrix.
  • Gwiritsani ntchito buffer yosefedwa mwatsopano (0.22 µm) kuti mupewe kuyambitsa tizilombo toyambitsa matenda.
  • Mizati ya qEV SMART imabwera yofanana mu PBS yosefedwa yomwe ili ndi <0.1% w/v sodium azide.

Kukhazikitsa Kolamu ndi Kulinganiza

1. Gwirizanitsani ndime ndi sampChosungiracho chiyenera kukhala mkati mwa kutentha kwa 18-24 ˚C.
   Chenjezo
   Osachotsa zipewa mpaka gawo litafika kutentha kwa ntchito.
   Sampma buffer ayenera kuchotsedwa mpweya komanso kutentha kwachipinda kuti pasakhale thovu la mpweya pabedi la gel osakaniza.
2. Ikani mzatiwo mowongoka pa choyikapo chokonzekera kugwiritsidwa ntchito. The Automatic Fraction Collector (AFC) ikupezeka ku store.izon.com
3. Tsukani posungiramo ndi buffer.
4. Musanalumikize chosungira ku ndime, onjezani 5 mL ya nkhokwe ku nkhokwe ndikudikirira kuti frit inyowe ndipo buffer iyambe kudutsa.
   • Ngati frit ikuchedwa kunyowa, ikani kukakamiza pamwamba pa chosungiramo ndi chikhatho cha dzanja lanu kuti muthandizire kuyenda.
5. Lolani kuti buffer igwire ntchito mpaka itayima pa frit.
6. Chotsani kapu cholumikizira ndime, onjezerani cholumikizira ndi chotchinga, ndikumangirira mwamphamvu (chisindikizo chabwino ndichofunikira) chosungira cholumikizira ku cholumikizira kusamala kuti musatseke thovu la mpweya mu cholumikizira.
7. Onjezani buffer ku posungira.
8. Chotsani kapu yapansi ndikulola kuti buffer iyambe kudutsa ndime.
9. Yambani ndime ndi gawo limodzi la sampndi buffer. Ngati chotchingira chotchinga china PBS ikugwiritsidwa ntchito, gwirizanitsani gawoli ndi magawo atatu a bokosi latsopanolo.
   Gwiritsani ntchito buffer yosefedwa mwatsopano (0.22 µm) kuti mupewe kuyambitsa tizilombo toyambitsa matenda.

Sampndi Loading

1. Kuti mupewe kutsekeka kwa zosefera, tikulimbikitsidwa kusefa kapena centrifuge biological sample kuchotsa zinthu zazikulu. Onani Gawo 4.1: Malangizo Ogwiritsira Ntchito Kuti mudziwe zambiri.
2. Pitirizani kulola kuti buffer idutse pamndandanda. Chigawocho chidzasiya kuyenda pamene buffer yonse yalowa muzitsulo zotsegula.
3. Kwezani okonzeka centrifuged samplembani voliyumu ku frit yotsitsa.
   Chenjezo
   Pewani kuyimitsa mzati pakuyenda kwa nthawi yayitali kuti muwonetsetse kulekanitsidwa kolondola kwa EV.
4. Nthawi yomweyo yambani kutolera voliyumu ya buffer (izi zikuphatikiza voliyumu yomwe idachotsedwa potsitsa sample).
5. Lolani sample kuthamangira mu column. Mzere udzasiya kuyenda pamene onse a sample walowa mu loading frit.
6. Kwezani mgawo ndi buffer ndikupitiliza kusonkhanitsa voliyumu ya bafa.
   ZINDIKIRANI
   Kuti mutenge ma voliyumu olondola, ingokwezani voliyumu yomwe ikufunika pamwamba pa ndime, dikirani kuti voliyumu idutse mpaka kutuluka kutayima ndikubwereza.
   Zosiyana samples angapereke zosiyana pang'ono elution profiles ndi chiyero, chifukwa chake kuyeza koyambirira kwa ndende ya EV ndi zowononga mapuloteni m'magulu osonkhanitsidwa ndikulimbikitsidwa.

Column Flush ndi Kusungirako

1. Zigawo zonse zomwe mukufuna zitasonkhanitsidwa tsatirani ndondomeko yoyeretsera yomwe yatchulidwa pambuyo pake, musanalowetse sample.
2. Ngati mukusunga mzati kuti mudzagwiritse ntchito mtsogolo, chitani njira yoyeretserayo ndi bafa yokhala ndi bacteriostatic agent (mwachitsanzo 0.05 % w/v sodium azide kapena 20% ethanol).
3. Mizati akhoza kusungidwa firiji pambuyo ntchito, kupereka
   ayeretsedwa molingana ndi malangizo omwe ali pamwambawa. Ngati njira zoyenera sizikupezeka ndiye kuti mizati imatha kusungidwa pa + 4 mpaka +8 ˚C mukatha kugwiritsa ntchito.

Kubwezeretsa Kuyenda Kwa Mzere Pambuyo Kutsekeka chifukwa cha Airlock mu Junction

1. Ikani kapu yapansi pamzati.
2. Chotsani posungira katundu.
3. Tsegulani kapu ya mzati ndikuwonjezera chotchinga pamwamba mpaka chotchingacho chikhale chofanana ndi m'mphepete mwapamwamba.
4. Limbitsaninso kapu yazazambiri pokakamiza kutchingira mmwamba podutsa polumikizira cholumikizira.
5. Onjezani 2 mL wa buffer ku posungira ndikulola kuti buffer idutse mpaka itayima pa frit.
6. Mosamala angagwirizanitse potsegula posungira kwa cholumikizira kusamala kupewa msampha aliyense thovu mpweya mu cholumikizira.
7. Onjezani chotchinga chowonjezera ku malo osungira musanachotse chipewa chapansi.
8. Mzere uyenera kuyambanso kuyenda.

ZOKHUDZA

Kuyeretsa Kolamu ndi Kuyeretsa
Tsukani ndi kuchotsa mapuloteni obwera, mapuloteni osamangika ndi lipoprotein potsuka ndimeyo ndi 100 ml ya bafa, kutsuka gawolo ndi 2 mL ya 0.5 M NaOH, kenaka yambani ndi osachepera 100 mL ya buffer mpaka pH ya chotchinga chochotsedwa. ndizofanana ndi pH yoyambira yosambitsira. PH ibwereranso ku ndale pambuyo pa magawo pafupifupi 2-3 a ma bafa ochapira.

Ma Protocols a EV Isolation from Common Sources
Onani Izon Support Center support.izon.com kuti mupeze zolemba ndi ma protocol wamba a EV samples. Ngati simukudziwa choti muchite kuti mukonzekere sample, chonde lemberani support@izon.com kuti awathandize.

EV Analysis Pogwiritsa Ntchito TRPS
Izon imalimbikitsa kusanthula kwa TRPS kuti mudziwe kukula kwa tinthu, kukhazikika, ndi kuthekera kwa zeta. Izon Reagent Kit imaphatikizapo njira zokutira zokutira pore, kuchepetsa kumangirira kosakhazikika komanso kumapereka kukhazikika kokhazikika komanso kolondola komanso kusanthula ndende.
Pakuwunika kwa TRPS kwa ma EV, Izon imalimbikitsa kuchepetsedwa koyamba kwa 1/5 kapena 1/10 mu electrolyte. Konzani dilution kuti mukwaniritse kuthamanga kwapamwamba kwambiri kwa pafupifupi 200 mpaka 1600 particles pamphindi kuti mupewe kutsekeka kwa pore.
Onani Izon Support Center support.izon.com kuti mumve zambiri pakuwunika kwa ma EV ndi TRPS.

www.izon.com

Zolemba / Zothandizira

IZON qEV2 Zowonjezera Ma Cellular Vesicles [pdf] Buku Logwiritsa Ntchito qEV2, Ma vesicles Owonjezera a Ma Cellular, Ma cell a Ma cell, qEV2, Ma Vesicles

Zothandizira

• Izon Science Safety Data Sheets (SDS)
• Izon Science Store
• Izon Science Support Center
• Dzipatulani & Muyese Zowonjezera Zowonjezera & Nanoparticles