IZON qEV10 Columns Isolate Extracellular Vesicles User Manual

Zigawo za IZON qEV10 Zimapatula Ma Vesicles Owonjezera

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Zogulitsa za Izon zidapangidwa ndikupangidwa pansi pa dongosolo labwino lovomerezeka ku ISO 13485:2016.
Zizindikiro zonse za chipani chachitatu ndi katundu wa eni ake.

Malingaliro a kampani 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 amasonkhanitsidwa basi mu chapakati chitsime. 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.

CHITETEZO NDI ZOOPSA

Onani ku Satifiketi Yachitetezo kuti mugawidwe ndikuyika zilembo zangozi ndi zidziwitso zofananira ndi chitetezo. The Safety Data Sheet ya qEV Isolation columns ili pa www.izon. com/zothandizira

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. NGATI WAKUMIRA: nthawi yomweyo imbani 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: www.izon.com/resources

Sodium azide imatha kupha munthu ikamezedwa kapena kukhudzana ndi khungu. Zitha kuwononga ziwalo zaubongo kudzera mukuwonekera kwa nthawi yayitali kapena mobwerezabwereza. Ndiwowopsa kwambiri ku zamoyo zam'madzi zomwe zimakhala 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 foni, kuyatsa cholumikizira magetsi, kapena kucheza ndi 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

qEV Size Exclusion Chromatography (SEC) mizati imalekanitsa tinthu tating'ono kutengera kukula kwake pamene akudutsa ndime yodzaza ndi porous, polysaccharide utomoni. Monga sampLe amadutsa mzati pansi pa mphamvu yokoka, tinthu ting'onoting'ono timalowa m'mabowo a resin popita pansi ndipo kutuluka kwawo pamzati kumachedwa (Chithunzi 1C). Monga sampLe amatuluka mzati, ma voliyumu otsatizana amasonkhanitsidwa. Tinthu adzagawidwa kudutsa mabuku kutengera kukula kwawo, ndi waukulu particles exiting ndime choyamba ndi zing'onozing'ono particles exiting ndime otsiriza.

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 utomoni wokhala ndi kukula kwa electron microscopic. (B) Chojambula cha sampmamolekyu akufalikira mu pores wa utomoni mikanda. (C) Kufotokozera mojambula za kulekana: (i) sample imayikidwa pakhoma; (ii) tinthu tating'ono kwambiri (chikasu) timachedwa kwambiri kuposa tinthu tating'onoting'ono (zofiira); (iii) tinthu tating'onoting'ono tating'ono tambiri tachotsedwa koyamba kuchokera 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 imapatula ma vesicles owonjezera kuchokera ku biological samples. Mizati ya qEV10 ili ndi tchipisi ta RFID kuti tigwiritse ntchito ndi Izon Automatic Fraction Collector (AFC). Ma chips awa sangakhudze kugwiritsa ntchito pamanja. Gawoli likuyenera kugwiritsidwa ntchito m'ma laboratories ofufuza ndi akatswiri kuti agwiritse ntchito kafukufuku wokha. Gawo la qEV silinapangidwe pofuna kufufuza matenda ndipo siliyenera kugwiritsidwa ntchito popanga zisankho zachipatala.
Mipingo ya qEV idapangidwa kuti izilekanitsa 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 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 akupezeka m'modzi mwa magawo awiri odzipatula, the
qEV/35nm mndandanda ndi mndandanda wa 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. Pakuti mulingo woyenera kuchira particles pakati 70 ndi 1000 nm ndi qEV/70nm mndandanda ndime tikulimbikitsidwa.

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

	qEV/35nm SERIES	qEV/70nm SERIES
Kukula kwa Particle (nm)	35 nm mpaka 350 nm	70 nm mpaka 1000 nm
Mulingo Wabwino Kwambiri Wobwezeretsa (nm)	Tinthu <110 nm	Tinthu > 110 nm

Zithunzi za qEV10

Gulu 4: Zofotokozera za qEV10

Dzinalo	qEV10
Zotsatizana	qEV10/35nm	qEV10/70nm
Kukula koyenera kolekanitsa	35-350 nm	70-1000 nm
Nthawi Yothamanga	~ Mphindi 40
Voliyumu yazanja	69.3 mL
Sampndi load volume	10 ml*
Mulingo woyenera wagawo	5 mL
Voliyumu ya buffer **	21.4 mL
Voliyumu yamphamvu	140 mL
Voliyumu yosonkhanitsidwa yoyeretsedwa **	20 mL
Chiwopsezo cha EV pambuyo pa voliyumu ya buffer **	10 ± 5 mL
Kutentha kogwira ntchito	18 mpaka 24 ºC
gawo lotetezedwa	PBS
Kukula kwakukulu kodutsa	1 μm
Pamwamba ndi pansi frit kukula	20 μm
pH yokhazikika yogwira ntchito	3 - 13
pH kukhazikika kuyeretsa-pamalo (CIP)	2 - 14
Nthawi ya alumali (ngati yasungidwa bwino)	miyezi 12

• 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. Kutsegula m'munsi sampma voliyumu amabweretsa kuchuluka kwa dilution ya sample. Mulingo woyenera bwino sample voliyumu yoyera pa qEV10
  ndi 10 mL.
• Makhalidwe a plasma ya anthuamples yekha.

qEV10 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. 4). Mapuloteni nthawi zambiri amatuluka pang'onopang'ono pamndandanda wa qEV/35nm. Mapuloteni apamwamba mu PCV makamaka chifukwa cha kuwonjezeka kwa mapuloteni omangidwa ndi EV.

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.

Chithunzi 3: Kuyerekeza kutulutsa kwa mapuloteni a plasma ndi kuchira kwa 69 nm liposomes pakati pa qEV10/35nm ndi qEV10/70nm.

Chithunzi 4: Kuyerekeza kwa plasma mapuloteni elution ndi kuchira milingo 200 nm particles pakati pa qEV10/35nm ndi qEV10/70nm.

qEV10 EV Elution Profile

Kutulutsa kwa ma vesicles nthawi zambiri kumafika pa 10 mL ± 5 mL pambuyo pa voliyumu ya buffer, kwa 10 mL s.ample voliyumu ndikutolera tizigawo ta 5 ml. Chithunzi 5 chikuwonetsa kutuluka kwa ma vesicles pamene 10 mL ya plasma sample imakwezedwa pagawo la qEV10/35nm.

Ma EV ambiri amachoka mu 20 ml pambuyo pa voliyumu ya buffer. Ngati chiyero chapamwamba chikufunika, sonkhanitsani 15 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 25 - 70 mL pambuyo pa voliyumu ya buffer. Ma vesicles aliwonse omwe adachira kupitilira 20 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 elution profile pa qEV10/35nm ndime yokhala ndi 10 mL ya plasma yodzaza; mapuloteni elute mochedwa kuposa vesicles extracellular (EVs) ndi ofanana kakulidwe particles> 60 nm. Kuchuluka kwa vesicle kunayesedwa pogwiritsa ntchito TRPS ndi mapuloteni okhudzana ndi mapuloteni ndi kuyamwa pa 280 nm.

qEV10 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 5mL ndi 10mL ya plasma. Onani kuchedwa kwa EVs mu 10 mL sample.

Mulingo woyenera bwino sample voliyumu yoyera pa qEV10 ndi 10 mL, zomwe zimapangitsa kuti ma vesicles alowe mu 20 mL PCV. Kutayika kwa ma vesicles kumachitika ndi sampKuchuluka kwa 10 mL kumawonjezera kuchuluka kwa vesicle. Ma EV omwe amasonkhanitsidwa kunja kwa 20 mL PCV savomerezedwa kuti afufuze kumtunda komwe kumayenera kukhala koyera kwambiri.

Chithunzi 6: Zotsatira zakukweza ma voliyumu akulu zimawonetsedwa mu ma vesicles akunja (EVs) ndi tinthu tating'onoting'ono > 60 nm pro.files kuchokera pakukweza 5mL ndi 10mL ya plasma.

Zotsatira za Mtundu wa Resin pa Protein Elution Profile
PCV pa qEV10 ili ndi mapuloteni ochepa kwambiri, ndipo mapuloteni amawonjezeka m'mavoliyumu apambuyo, monga momwe tawonetsera pa Chithunzi 7. Mapuloteni apamwamba a qEV10 / 35nm amayamba chifukwa cha kuchira kwakukulu kwa ma EV ang'onoang'ono mu PCV (onani mkuyu. 2).

Chithunzi 7: Peresentitagndi mapuloteni cf. kuyambira kuchuluka kwa mapuloteni kuchokera pakukweza 10 mL ya plasma pa qEV10. Mipiringidzo ya pinki ndi PCV. Mipiringidzo ya qEV10/70nm ndi yapinki yoderapo komanso yabuluu wakuda. Mipiringidzo ya qEV10/35nm ndi yowala pinki komanso yopepuka yabuluu.

MALANGIZO OGWIRITSA NTCHITO MANUKO

Gawo lotsatirali likupereka malangizo ogwiritsira ntchito mizati ya qEV pamanja. Kuti mugwiritse ntchito mizati ya qEV 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 likuyenda bwino:

• Centrifuge samppang'ono musanalowetse pa column. Kupewa kutsekeka kwa frits, 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 macrovesicle, gwiritsani ntchito mphamvu zochepa za g pa gawo lachiwiri la 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, 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.
  • 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 qEV single, 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 kuipitsidwa.
  • Mizati ya qEV 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.
   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.
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. Ngati mukugwiritsa ntchito PBS ngati buffer yanu ndiye kuti gawo lanu lakonzeka kugwiritsidwa ntchito. Ngati ntchito yanu yotsika pansi yakhudzidwa ndi 0.05% sodium azide, tsitsani gawoli ndi magawo awiri a PBS. Ngati chotchingira chotchinga china kupatula PBS ndichoti chigwiritsidwe ntchito, gwirizanitsani gawoli ndi magawo atatu a bafa atsopanowo.

Gwiritsani ntchito buffer yosefedwa mwatsopano (0.22 µm) kuti mupewe kuyambitsa tizilombo toyambitsa matenda.

Sampndi Loading

1. Kupewa kutsekeka kwa frits, 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.
   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.
7. Voliyumu ya buffer ikasonkhanitsidwa, pitilizani kusonkhanitsa Purified Collection Volume (PCV).
   Kuti mutenge ma voliyumu olondola, ingokwezani voliyumu yomwe ikufunika pamwamba pa ndime, dikirani kuti voliyumu idutse mpaka kutuluka kutayima ndikubwereza.

Column Flush ndi Kusungirako

1. Tizigawo tomwe tikufunikira titasonkhanitsidwa, mzatiwo uyenera kutsukidwa ndi kuyeretsedwa kuti uchotse mapuloteni otsalira. Tsukani gawolo ndi 140 mL ya buffer mwachindunji mukamaliza kusonkhanitsa tizigawo, kenako yambani ndimeyi ndi 5 ml ya 0.5 M NaOH, kenaka yambani ndi 140 mL ya buffer kuti mubwezeretse pH yanthawi zonse musanalowetsenso s.ample.
   Kungothamangitsa ndi buffer yochuluka pambuyo posonkhanitsa tizigawo sikokwanira kuyeretsa gawolo kwathunthu ndipo pakhoza kukhala zopititsira patsogolo kuchokera m'magawo am'mbuyomu.amples.
2. Ngati mukusunga mzati kuti mugwiritse ntchito mtsogolo, ziyenera kusungidwa mu bacteriostatic agent monga PBS yomwe ili ndi 0.05% w/v sodium azide, kapena 20% ethanol. Mizati yosungidwa mu 20% ethanol iyenera kutsukidwa ndi magawo awiri a madzi a DI mutatha kutsukidwa, kenako ndikuthira ndi magawo awiri a 20% ethanol kuti musungidwe. Mizati yosungidwa mu nkhokwe iyenera kutsukidwa ndi magawo awiri a bafa.
   Pewani kuwonjezera 20% ethanol kuti ikhale mkati mwa mzati chifukwa izi zimatha kuyambitsa mchere mkati mwa bedi la gel ndikuwononga gawolo.
3. Mipingo imatha kusungidwa kutentha kwa chipinda mukatha kugwiritsidwa ntchito, malinga ngati yatsukidwa molingana ndi malangizo omwe ali pamwambapa. 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

Protocols for 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. The Izon TRPS Reagent Kit imaphatikizapo njira zokutira zomatira pore, kuchepetsa kumangirira kosakhazikika komanso kumapereka kukhazikika komanso kolondola komanso kusanthula ndende.
Pakuwunika kwa TRPS kwa ma EV, Izon amalimbikitsa kuchepetsedwa koyambirira 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.

Zolemba / Zothandizira

Zigawo za IZON qEV10 Zimapatula Ma Vesicles Owonjezera [pdf] Buku Logwiritsa Ntchito qEV10, Mizati Isolate Extracellular Vesicles, Isolate Extracellular Vesicles, Extracellular Vesicles, qEV10, Vesicles

Zothandizira

• Izon Science Support Center
• Dzipatulani & Muyese Zowonjezera Zowonjezera & Nanoparticles
• Zothandizira kuphatikiza Maupangiri Ogwiritsa, SDS, Brochure ndi Zolemba za App