dynamic BIOSENSORS heliX cyto Fully Automated Laboratory Analysis System

Zofotokozera

• Dzina lazogulitsa: heliX cyto
• Wopanga: Malingaliro a kampani Dynamic Biosensors GmbH
• Mtundu: 1.0

Zambiri Zamalonda

• Dongosolo la heliX cyto lapangidwa kuti likhale limodzi la cell Interaction Cytometry (scIC) kuti lizitha kuyeza ma kinetics omangika a mamolekyu otchedwa fluorescently to targets on cell surfaces in the time-related .

Malangizo Ogwiritsira Ntchito Zogulitsa

Kugwiritsa ntchito heliXcyto Chip

• Chip cha heliXcyto chili ndi njira imodzi ya microfluidic yokhala ndi madontho a electrode yojambula ndi kuyeza. Onetsetsani kuyika bwino kwa chip ndikutsatira malangizo otsekera ma cell.

Kusamalira Chip

• Gwiritsani ntchito Chip cha Maintenance poyeretsa, kuyesa, ndi kukonza ntchito zokha. Pewani kugwiritsa ntchito poyesera kuti mupewe kuipitsidwa.

Kuthamanga kwa Buffer

• Gwiritsani ntchito Running Buffer 1 (RB 1) poyezera. Onani tsamba lofananira la scIC mu bukhuli kuti mumve zambiri.

Sensorgram Analysis

• Yang'anirani sensorgram mu nthawi yeniyeni mu pulogalamu ya heliOS panthawi yoyezera. Unikani mayankho a fulorosenti pakapita nthawi kuti mutenge mitengo ya kinetic.

Chisangalalo

• Ganizirani kugwiritsa ntchito passivation ngati sitepe yosankha kuti mupewe kutsatsa kwa analyte poyimitsa chochitira zinthu pa chip.

Kukhazikika

• Normalization ndi njira yosinthira deta kuti ifanane ndi kusanthula. Tsatirani ndondomeko za normalization malinga ndi buku la ogwiritsa ntchito.

MAU OYAMBA

• Bukuli limatanthauzidwa ngati lalifupiview za dongosolo la heliXcyto ndi luso lake. Mitu yonse ikuwonjezedwa mu kalozera wamkulu wa heliXcyto wopezeka pa https://www.dynamic-biosensors.com/helios-download-latest/

HeliXcyto Terminology single cell Interaction Cytometry

• single-cell Interaction Cytometry (scIC) ndiukadaulo womwe umayesa ma kinetics omangika a molekyulu yolembedwa ndi fluorescently (analyte) kupita ku chandamale (ligand) pamtunda wa cell pojambula chizindikiro cha fulorosenti munthawi yotsimikizika.

Analyte

• Analyte ndi molekyulu yolembedwa ndi fulorosenti yomwe imatha kumangirira chandamale pamwamba pa selo.

Ligand

• Ligand” ndi dzina lomwe limagwiritsidwa ntchito mu pulogalamu ya heliOS kufotokoza chandamale pa cell pomwe wosanthula amatha kumangirira.Izi zitha kukhala molekyulu ya cholandilira, lipid, shuga kapena molekyulu ina yapa cell.

Cell Trap

• Misampha ya m'maselo ndi yotha kupindika, yolumikizana ndi ma polima makola pa chip heliXcyto. Amapangidwa kuti azigwira ndi kusasunthika ma cell akulu akulu kuchokera pafupifupi 6 mpaka 25 µm munjira yoyenda. Misampha yama cell imapezeka mumitundu itatu: yaying'ono, yapakatikati ndi yayikulu.

heliXcyto Chip

• Tchipisi za heliXcyto zili ndi njira imodzi ya microfluidic yokhala ndi zotseguka mbali zonse. Mawanga awiri agolide a elekitirodi ali pakati pa njira.
• Malo amodzi amakhala ngati malo owonetserako, ndipo malo achiwiri amanyamula 3D biocompatible polima makola kuti agwire ma cell ndipo amakhala ngati muyeso. Malo oyezera amatha kukhala ndi misampha 1 kapena 5 yamtundu womwewo.
• Malo owonetsera amalola kuloza zenizeni zenizeni za chizindikiro cha fulorosenti. Chip cha heliXcyto ndi chogwiritsidwanso ntchito komanso chotaya.

Kusamalira Chip

• Maintenance Chip ndi njira imodzi ya microfluidic chip yomwe imagwiritsidwa ntchito poyeretsa, kuyesa ndi kukonza ntchito zonse.
• Zochita izi zikuphatikiza Kuyeretsa & Kugona chizolowezi, Kudzuka ndi Prime routine, Kusamba kwa System ndi mayeso a Fluidics. Chip ichi sichiyenera kugwiritsidwa ntchito poyesera zenizeni ndi mayankho a cell/mapuloteni kuti ateteze kuipitsidwa kwina kwa chip.

Kuthamanga kwa Buffer

• Running buffer ndi buffer yomwe imagwiritsidwa ntchito mu heliXcyto pakuyezera. Kugwiritsa ntchito Running Buffer 1 (RB 1) kumalimbikitsidwa.
• Chonde onani tsamba lofananira la scIC lomwe lili mu bukhuli kuti mudziwe zambiri.

Sensorgram

• Sensa ya scIC ndi chiwembu cha mayankho a fluorescence m'mawerengedwe pamphindikati (cps) pakapita nthawi, kusonyeza kupita patsogolo kwa kuyanjana pamaselo kapena m'maselo. Mzerewu ukhoza kukhala viewed mu nthawi yeniyeni mu heliOS panthawi yoyezera.
• Mukasanthula ma sensagalamu, kuwonekera koyenera koyenera kumayendedwe a fulorosenti kumatsimikiziridwa ndi kuchuluka kwa kinetic (kon, koff) kuchotsedwa.

Chisangalalo

• Passivation ndi sitepe yosankha pothamangira, pomwe reagent passivation imabayidwa pa chip ndikumakulitsidwa kuti atseke. Izi zitha kuthandiza kupewa kutsatsa kwa ma analytes ku zida za chip.

Kukhazikika

• Gawo lokhazikika limagwiritsidwa ntchito powerengera kusiyana pang'ono kwa ma siginoloji chifukwa chosonkhanitsira ma siginecha kuchokera pamalo aliwonse oyezera ndi chowunikira chosiyana. Utoto wa fulorosenti wokhazikika (kutengera kuchuluka kwa ma analyte ndi digiri ya zolemba za analyte) umabayidwa kumayambiriro kwa kuyesa. Gawo lokhazikikali limaphatikizidwa ndi njira yoyezera, ndipo nsonga yoyezera yokhazikika imagwiritsidwa ntchito kuwongolera zokha kusiyana pakati pa zowunikira pakusanthula deta, isanatchulidwe nthawi yeniyeni.

Mfundo Zoyezera za scIC

Mapulogalamu a scIC

• single-cell Interaction Cytometry (scIC) imayesa milingo ya kinetic ndi kugwirizana kwa analyte otchedwa fluorescently analyte kumanga ndi kumasula kuchokera ku cholinga chake mwachindunji pa maselo amoyo.
• Kuzindikira ma analytes mu scIC sikudalira kukula kotero ndikofunikira ku molekyu iliyonse kuchokera ku sub-nm kupita ku> 100 nm ndipo imagwirizana ndi magulu onse a molekyulu kuchokera ku mamolekyu ang'onoang'ono, ma peptides, ma aptamers, nanobodies, affibodies, ma antibodies, mawonekedwe a antibody bispecific, mapuloteni, ma protein complexes, lipide vesicles, mpaka ma virus

ukadaulo wa scIC utha kuthana ndi madera otsatirawa:

• kuyeza kwa chizindikiro ampzowerengera m'mawonekedwe omanga
• Kinetic kusanthula kwa kon ndi koff mitengo
• kuwunika kogwirizana ndi kulimba mtima kudzera mumitundu ya koff ndi biphasic fit
• mayeso enieni
• wachibale quantification wa nembanemba mapuloteni pa selo pamwamba
• maphunziro a epitope binning ndi mpikisano
• zoletsa zoletsa
• kuwunika kwa mkati mwa mapuloteni omwe akuwunikira

heliXcyto Hardware Kutha

Fluidic System

• Dongosolo la fluidic la heliXcyto limadyetsedwa ndi mabotolo amtundu wa 3, zomwe zimapangitsa kuti pakhale kusiyanasiyana koyendetsa ndi kukonza. Mapampu mu heliXcyto amatha kukhazikitsidwa kuti aziyenda pakati pa 20 ndi 500 µL/mphindi, kutengera magawo osiyanasiyana.ample ndi zofunikira za assay. Njira yothamanga ya chip imalumikizana ndi dongosolo la fluidic kudzera m'mipata iwiri. Kutsegulira kumanzere kumalumikizidwa ndi sample, buffer, ndi mizere yochapira, pomwe kutsegulira kumanja kumalumikizidwa ndi mzere wosinthika.
• Dongosolo lanjira ziwiri la fluidic limathandizira kugwidwa kwa cell ndikuyendetsa masitepe osiyanasiyana oyezera ndipo pamapeto pake kusinthika kwa chip kwa in-assay chip reusability.
• Chithunzi 1. Kuphatikiza kwa Chip mu Fluidic system

Optical System

• Chizindikiro cha fluorescence chimasangalatsidwa ndi ma diode ofiira ndi obiriwira otulutsa kuwala (ma LED) ndipo amadziwidwa ndi zowerengera zinayi za mafotoni amodzi, omwe amasonkhanitsa zizindikiro zofiira ndi zobiriwira kuchokera pamalo aliwonse akuya kwathunthu kwa tchanelo.
• Zizindikiro ziwiri zodziyimira pawokha zitha kuyang'aniridwa nthawi imodzi pamalo oyezera, kulola kuyanjana kuwiri kuti ayezedwe nthawi imodzi munjira yoyeserera yamitundu iwiri.
• Chithunzi 2. Kupanga kwa Optics
• Kutoleredwa kwa ma sign kuchokera pamalo aliwonse oyezera kumayendetsedwa ndi chowunikira chosiyana, zomwe zingapangitse kusiyana pang'ono paziwerengero zosaphika. Kuti tichite izi, gawo lokhazikika limaphatikizidwa muzoyesa zopanga deta.
• Kuphatikiza pa zowerengera za chithunzi chimodzi, chidacho chili ndi kamera ya CCD ndi microscope yowunikira. Zida izi zimalola kujambula kwa nthawi yeniyeni ya maselo, zomwe zimathandizira kuwunika kwa kukhulupirika kwa ma cell ndikuwunika momwe mumayezera.
• Kukonzekera kophatikizanaku kumatsimikizira kuti zochitika zonse ndi zochitika zamoyo zimatsatiridwa, kupereka kuwunika kwatsatanetsatane kwa kuyesa.

scIC ntchito

Njira Yoyezera ya scIC ikhoza kugawidwa m'magawo atatu

1. Mapangidwe Oyesera mu heliOS: Muyeso uliwonse umayamba ndikupanga kuyesa mu pulogalamu ya heliOS. Mayendedwe a assay amakhazikitsidwa posankha njira zodziwikiratu ndikusintha magawo oyesera, monga ma cell omwe amagwiritsidwa ntchito, kuwunika kwa analyte, ndi mikhalidwe ya buffer. heliOS imawerengera yokha kuchuluka kwa maselo, ma analyte, ma buffers, ndi mayankho ena ofunikira pakuyezera, ndikuwongolera njira yokonzekera.
2. Kukonzekera kwa ma buffers, analytes, ndi ma cell: Pambuyo poyeserera kumalizidwa, zida zofunikira zimakonzedwa molingana ndi zomwe zaperekedwa ndi heliOS. Ma buffers, ma analyte, ndi ma cell amalowetsedwa mu makina opangira zidaamplero.
3. Kuyeza ndi Kusanthula Deta: Dongosololi limapanga miyeso yokhazikika yoyezera nthawi yeniyeni yomwe imafuna kuti wogwiritsa ntchito asalowererenso. Deta ikhoza kuwunikidwa pamene muyeso ukugwirabe ntchito kuti mudziwe zambiri za zotsatira.

scIC Mayeso mu heliOS

Kuphatikiza pa kuyesa kopanga deta, pulogalamuyi imaperekanso njira zoyesera chip, kuyeretsa ndi kukonza zida.

Table 1. Mayesero Otsimikizika Opanga Deta

ScIC Kinetics	Imayesa ma kinetics athunthu pama cell omwe ali ndi ma analyte mayanjano osinthika pakati pa 1 ndi 5, ndikutsatiridwa ndi kupatukana kumodzi pambuyo pakukhazikika kwambiri. Njira yobiriwira kapena yofiira. Lili ndi njira yoyesera ya Analyte yokha.
scIC Kinetics - Mtundu Wapawiri	Imayesa ma kinetics athunthu pama cell omwe ali ndi ma analyte mayanjano osinthika pakati pa 1 ndi 5, ndikutsatiridwa ndi kupatukana kumodzi pambuyo pakukhazikika kwambiri. Zonse zobiriwira ndi zofiira zimayatsidwa nthawi imodzi. Lili ndi mwayi woyeserera kokha wa Analyte.
scIC Dissociation - Mtundu Wapawiri	Imayesa kudzipatula kwa analyte kuchokera ku maselo omwe adayikidwa kale ndi analyte otchedwa fluorescently ndi njira yobiriwira ndi / kapena yofiira.

Kukonzekera ndi Kupititsa patsogolo Kuyesa kwa scIC Measurement

Pali mfundo zingapo zomwe muyenera kuziganizira musanayike muyeso wa scIC

• Analyte concentration, Degree of Labeling, katundu, ndi mtundu.
• Yankho la Normalization ndi mphamvu yosangalatsa.
• Ubwino wa ma cell ndi kachitidwe.

Analyte Concentration ndi Digiri ya Kulemba

• Malinga ndi machitidwe oyezera a kinetic, timalimbikitsa kusankha mtundu wa analyte molar concentration wa 0.1x mpaka 10x woyembekezeka woyembekezeka wa dissociation constant (Kd) wa ma kinetics angapo. Pamiyezo ya kinetics yokhazikika kapena yodzipatula, kuchuluka kwa analyte kuyenera kukhala pakati pa 1x ndi 10x Kd yomwe ikuyembekezeka. Voliyumu ya analyte yofunikira yowerengedwera kwa nthawi yolumikizana ndi mphindi 10 pakuyenda kwa 25 µL/mphindi pafupifupi pafupifupi 300 µL.
• Degree of Labeling (DOL) ya owerengera ayenera kukhala 1, ngakhale ma DOL a 2 kapena 3 akadali ovomerezeka. Komabe, dziwani kuti kuchuluka kwa zilembo pa analyte kumatha kukhudza momwe amamangirira, ndipo zitha kukulitsa mwayi wophatikiza kapena kumanga mosadziwika bwino. Kuti mukwaniritse DOL yabwino, tsatirani ndondomeko yoperekedwa ndi Zolemba Zolemba zomwe zili ndi utoto wofiira kapena wobiriwira kuchokera ku mzere wa heliXcyto reagents.
  Kukhazikika
• Normalization ndi sitepe yoyamba mu njira zonse zopangira deta. Imalipiritsa kusinthasintha kwakung'ono kwa ma siginecha komwe kumachitika pogwiritsa ntchito zowunikira zapadera pagawo lililonse loyezera. Mu sitepe iyi, utoto wa fulorosenti pamtunda wotchulidwa ndi wogwiritsa ntchito umayikidwa kumayambiriro kwa kuyesa.
• Kuchulukira kwa Normalization Solution kumawerengeredwa pochulukitsa kuchuluka kwa analyte komwe kumagwiritsidwa ntchito poyeza ndi analyte's Degree of Labeling (DOL). Kuphatikizika uku kumawongolera mphamvu yachisangalalo ya LED yomwe imagwiritsidwa ntchito poyezera. Cholinga chake ndi chakuti Normalization Solution pachimake chifikire pafupifupi chizindikiro cha fulorosenti amplitude ngati ndende yapamwamba kwambiri ya analyte.
• Onani tchati cha Normalization solution mu bukhuli kuti muyike zoyambira. Zindikirani kuti tchatichi chimatsogolera zoikamo zoyambira, koma mphamvu yachisangalalo ya LED ikhoza kusinthidwanso pakukulitsa kuyesa.

Ubwino wa Maselo ndi Kukonzekera

• Ma cell akuyenera kukhala opitilira 95%, ndipo ma cell amayenera kukhala abwino. Kwa ma cell omwe amatsatira, timalimbikitsa kugwira ntchito ndi ma cell omwe ali olumikizana 50-70%.
• Kwa maselo oyimitsidwa, timalimbikitsa kukolola pakatikati pa chipika cha kukula. 50 µl ya kuyimitsidwa kwa selo pa 1E + 06 maselo / mL ndiyofunikira pakuthamanga.

Kuyimitsidwa maselo kukolola

1. Centrifuge pafupifupi. Ma cell a 2E + 06 pa 300 g kwa mphindi 7 kuti achotse media media. Taya zauzimu.
2. Tsukani ma cell kamodzi poyimitsanso cell pellet pafupifupi. 1 ml ya DPBS. Centrifuge kuyimitsidwa pa 400 g kwa mphindi 5 kuti pellet amoyo maselo. Mosamala kutaya supernatant kuchotsa zidutswa ndi TV.
3. Imitsaninso pang'onopang'ono mu 1 mL ya DPBS, sinthani kuyimitsidwa pamwamba pa cell strainer ndikuumitsa kuyimitsidwa ndi mphamvu yokoka.
4. Yezerani kuchuluka kwa ma cell a kuyimitsidwa koyimitsidwa ndikusintha kukhala 1E+06 cell/mL.
   • MFUNDO: Maselo ena oyimitsidwa amatha kupanga masango. Imitsaninso magulu pang'onopang'ono ndikuphatikiza sitepe yopumira musanayambe sitepe yoyamba ya centrifugation.
   • Gwiritsani ntchito nsonga za pipette zotambalala pamene mukugwira kuyimitsidwa kwa ma cell kuti mupewe kumeta ubweya wambiri panthawi yosinthira kapena kuyimitsanso.

Otsatira maselo kukolola

1. Tsukani ma cell ndi DPBS wosabala.
2. Dulani ma cell kuchokera mu botolo lokulitsa pogwiritsa ntchito njira yomwe mumakonda yodzipatula (mwachitsanzo TrypLE).
3. Imitsaninso ma cell osiyanitsidwa kumtundu womwe mumakonda wa media ndikusefa ndi 30 nm cell strainer.
4. Mwachidziwitso, onjezani EDTA ngati pali ma cell omwe amamatira kwambiri (5 mmM final concentration).
5. Centrifuge ma cell pa 300 g kwa mphindi 7 kuti muchotse media media. Taya zauzimu.
6. Imitsaninso maselo ku 1 mL ya DPBS.
7. Yezerani kuchuluka kwa maselo ndikusintha kukhala 1E+06 ma cell/mL.
   • MFUNDO: Ma cell media ochepetsedwa 1:4 ndi DPBS atha kugwiritsidwa ntchito kuwonjezera zomanga m'ma cellampkuchepera ngati ma cell tcheru kapena kuyeserera kwanthawi yayitali.

Kukonza Maselo

1. Centrifuge mpaka 10E + 06 ma cell pa 300 g kwa mphindi 7 kuti muchotse media media. Taya zauzimu.
2. Imitsanso cell pellet mu 1 mL PBS, centrifuge ndikutaya zamphamvu.
3. Imitsaninso pellet ya cell mu 500 µL PBS/2% PFA (62.5 µL ya 16% PFA yankho + 437.5 µL PBS).
4. Ikani ma cell pa kutentha kwa mphindi 10 (ndikugwedeza pang'ono pang'ono).
5. Onjezani 500 µL ya PBS ndi centrifuge kuyimitsidwa pa 400 g kwa mphindi 5 kuchotsa PFA.
6. Taya zauzimu.
7. Imitsaninso ma cell mu 1 mL PBS, sefa kudzera pa 30 µm cell strainer, centrifuge (400 g, 5 min) ndikutaya zamphamvu.
8. Imitsaninso ma cell pafupifupi 1 mL ya PBS (posankha: sinthani mpaka ndende yomaliza ya 5E+06 cell/mL).
9. Sungani maselo okhazikika pa 2-8 ° C, gwiritsani ntchito mkati mwa mwezi umodzi.
   • Ngati ma cell ambiri asinthidwa, chonde onjezerani ndalama zonse molingana.
   • ZINDIKIRANI: Kuthamanga kwa centrifugation panthawi yokolola ma cell kumatha kusinthidwa kukhala mtundu wa ma cell, ngati ma cell ali tcheru komanso mphamvu zotsika za g-mphamvu zimagwiritsidwa ntchito.
   • Kukhazikika kwa PFA kumatha kukhala kosiyana pakati pa 0.5 ndi 4%.

scIC Assay kukhazikitsa

Mayesero otsatirawa a scIC ayenera kuphatikizidwa pakukula kwa mayeso ndipo atha kubwerezedwa mkati mwamayesero oyeserera pambuyo pake.

Kukula kwa ntchito ya assay kumagawidwa m'magawo atatu otsatizana:

1. Cyto chip test
2. Analyte mayeso okha
3. Kinetics kuyesa

Cyto Chip Test

• Ubwino wa chip chatsopano uyenera kuwunikiridwa musanagwiritse ntchito cyto chip muyeso. Yambani ndikuyesa Cyto Chip Test padera. Onani mutu wa Helix cyto chip mu kalozera wamkulu wa momwe mungakhazikitsire ndikuwunika kuyesa kwa chip.

Analyte Only Test

• Kukula kwa scIC assay kumayamba ndi Analyte Only Test, yomwe imayesa machitidwe a owunikira olembedwa ndi fluorescent pa chip chatsopano popanda ma cell.
• Mayesowa amathanso kubwerezedwa nthawi ndi nthawi kuti awone kukhazikika kwa osanthula olembedwa. scIC Analyte Only Test itha kukhazikitsidwa muzoyesa za Kinetics poyambitsa bokosi la Analyte lokha pansi pa zoikamo za Cell.
• Pakuwunika kwaubwino wa analyte, mayendedwe ogwiritsidwa ntchito kwambiri ndi okwanira ndipo amatha kukhazikitsidwa poletsa onse koma amodzi mwa mayanjano omwe akuyesa.

Kinetics Assay Workflow Setup

• Ma kinetics assay nthawi zambiri amagwiritsidwa ntchito mkati mwa kayendedwe ka ntchito komwe kumakhala ndi zoyeserera zomwe zimapanga deta komanso zokonza. Wowunikirayo akadutsa mayeso owongolera a Analyte okha, amatha kugwiritsidwa ntchito muyeso pama cell.
• Kuti muwerenge kuchuluka kwa kinetic modalirika, kuyankha kwa ma siginecha panthawi yolumikizana kuyenera kudalira ndende, kuchulukirachulukira ndikuwunika kwambiri.
• Kuyika kwakukulu kuyenera kufika pamtunda, zomwe zimasonyeza kuti kukhazikika kwachitika. Kupatukana kuyenera kuyendetsedwa motalika kokwanira kuti kumasule gawo lalikulu la analyte omangika (kuposa 5%) kuti athe kukhazikika kodalirika pamapindikira.
• Gwiritsani ntchito magawo osasinthika pazoyeserera ngati poyambira ndikusinthanso kutengera zotsatira zanu.

Example 1. Njira yoyeserera yovomerezeka

Mayendedwe oyeserera amalola ogwiritsa ntchito kuyika pamzere njira zoyezera ndi kukonza kutengera zosowa zawo zoyeserera.

Njira yoyeserera ikhoza kukhala ndi njira zotsatirazi mu dongosolo lomwe lasonyezedwa:

1. Prime (Chip chokonzekera)
2. scIC Kinetics (Maselo A, Analyte A, Chip Cyto)
3. Kusambitsa System (Chip chokonza)
4. scIC Kinetics (Maselo B, Analyte A, Chip Cyto)
5. Kusambitsa System (Chip chokonza)
6. Sinthani scIC Kinetics yokha (Analyte A, Cyto chip)
   • Masitepe a Prime ndi System Wash amachitidwa pa Chip Maintenance. Kusamba Kwadongosolo kumaphatikizidwa mukamasinthira ku mzere wina wa cell komanso musanachite mayeso a Analyte Only kumapeto kwa ntchito.
   • Kuti mudziwe zambiri, onani gawo la Kusamba kwa Cyto mu bukhuli.
   • Mukayika ma kinetics pama cell, lingalirani momwe ma cell amakhalira, zomwe zimadalira mzere wa cell.
   • Pamizere yovuta ya ma cell, tikulimbikitsidwa kuti muyese mayeso a 1-2 pamayendedwe oyeserera. Kuti mupeze ma cell olimba kwambiri, zoyeserera zowonjezera zitha kuimitsidwa.
   • ZINDIKIRANI: Kuti mudziwe zambiri, konzekerani njira yothetsera ma cell m'mabotolo osiyana potchula maselo mosiyana.
   • Onetsetsani kuti ma reagents onse ndi atsopano ndikusefedwa kudzera mu fyuluta ya 0.22 µm. Pomaliza, ikani ma reagents okonzeka mu chida monga mwalangizidwa ndi heliOS.
   • Kuthamanga kutha kuyambika podina chizindikiro cha buluu "Run" ndikutsata njira za Assay Start Wizard.
   • Kuthamanga kukangoyamba, chipangizocho chimagwira ntchito palokha mpaka ntchito yonse yoyeserera itatha.

Kusanthula kwa kuyesa kwa scIC

ScIC Analysis Ntchito Yoyenda

• Deta ya scIC ikhoza kusiyana ndi deta yodziwika bwino ya biosensor chifukwa cha zovuta zachilengedwe zomwe zimachitika pa maselo ogwidwa pa heliXcyto chip pamwamba, zomwe zingayambitse zolakwika za sensorgram.
• Chifukwa chake, kuwongolera kwaubwino kumatsindikiridwa pazithunzi zojambulidwa pagawo lililonse loyezera komanso pamasensa opangidwa patsamba lofikira la kusanthula kodzichitira.
• Kuphatikiza apo, heliOS imapereka zida zothanirana ndi zolakwika za sensorgram panthawi yosanthula deta ya scIC.

scIC data analysis process:

1. Kuyang'ana Zithunzi:
   • Yang'anani zithunzi zonse zojambulidwa mumiyeso yonse (Zithunzi tabu pazenera loyesera).
   • Ndi ma cell angati omwe adakhalabe okhazikika mumisampha munthawi yonseyi?
   • Kodi ma cell ali bwanji? Yang'anani granularity ndi cell integrity.
   • Kodi misampha imakhala yoyera pambuyo pa sitepe yokonzanso?
2. Kufufuza kwa Raw Data:
   • Yang'anani zomwe zasungidwa pa malo 1 ndi malo oyezera 2
   • Chizindikiro chapamwamba chokhazikika chiyenera kukhala pafupi kapena chokwera kuposa chizindikiro chapamwamba kwambiri cha data.
   • Kodi siginecha imabwereranso pamalo oyambira pamagawo onse awiri, kapena pali umboni wakumangirira kosadziwika bwino?
3. Kufufuza Kwazokhazikika:
   • Kodi ma jakisoni amadumphira pa malo 1 ndi malo 2 akukuta bwino?
4. Kufufuza kwa Data Yotchulidwa:
   • Kodi sitepe yokonzanso ikubweretsanso chizindikiro pa chiyambi? Ngati sichoncho, fufuzani ngati pali maselo kapena zinyalala mumisampha pambuyo pa kubadwanso mwatsopanoviewndi zithunzi.
   • Kodi pali ma spikes, ma outliers, kapena zolakwika zina pama curve omwe atchulidwa? Ngati ndi choncho, review zithunzi kuti azindikire zomwe zingayambitse ndikuganizira zosintha pamanja.
5. Kufufuza kwa Data Fit:
   • Kuyesa kuwunika kowoneka bwino
   • Kodi kukwanirako kumafotokoza bwino momwe ma curve, kapena mzere wokwanira umadutsa sensorgram?
   • Kodi kukwanirako kumafotokoza moyenerera kupindika kwa data?
   • Ndi ampzikugwirizana ndi deta yotchulidwa?

scIC Automated Data Analysis

• Kusanthula kodzipangira kwa scIC kumayendetsa deta yaiwisi kumagawo owongolera bwino ndikuyika deta ndikudina pang'ono.
  1. Tsegulani zoyeserera za scIC podina kawiri pazoyeserera zomwe zasankhidwa mu List Experiment List
  2. Dinani batani lalikulu la buluu Yendani pansi pa tabu yoyesera.
  3. Kuchokera kumayendedwe oyesera, sankhani kuyesa komwe mukufuna kusanthula.
  4. Sankhani Kinetics scIC kuchokera ku mitundu yowunikira yomwe ilipo ndikudina Next.
     • ZINDIKIRANI: Ngati kuyesaku kukuchitikabe, zoyeserera zomalizidwa zokha zidzawonetsedwa pamndandanda. Kuyesa kukasankhidwa, zenera lotsatira lidzawonetsa mitundu yonse yowunikira yomwe ilipo yokhudzana ndi njira / kuyesa kogwiritsidwa ntchito.
  5. Ngati pakufunika, konzani kusanthula pazenera lotsatira. Mchitidwe wofikiratu ndi "Kinetics - Free end level" ndi bokosi loyang'ana "Limbikitsani gawo lofikira ku Zero" loyatsidwa. Patukani pachitsanzo ichi pokhapokha ngati biology kapena deta ikufuna kufotokozera zovuta kwambiri.
  6. Kukonzekera kukamalizidwa, dinani Usanthuleni kuti muwone zithunzi zonse zotengedwa, ma sensorgram aiwisi ndi osinthidwa, ndi ma kinetics.
• Chifukwa cha zovuta za sampZosagwiritsidwa ntchito poyesa ma scIC, ma sensorgrams amatha kukhala ndi zolakwika.
• Kuti athane ndi izi, kuwongolera bwino kwa zithunzi ndi ma sensorgram kumagogomezeredwa pakuwunika kodzichitira.
• Ngati kuwongolera pamanja kapena kusanthula mozama kumafunika, zida zowongolera zidaperekedwa mkati mwa kusanthula kwamanja kwa Scratchpad.

heliXcyto Maintenance ndi Decontamination

• Kukonzekera kwa heliXcyto ndikofunikira kuti zitsimikizire kuti chidacho chimagwira ntchito bwino komanso moyo wautali. Kusamalira nthawi zonse kumaphatikizapo njira zoyeretsera zomwe zimalepheretsa kuipitsidwa ndi kusunga umphumphu wa dongosolo, zomwe zimapangitsa zotsatira zolondola komanso ntchito yabwino. Chisamaliro chokhazikika ndi chofunikira kuti chidacho chikhale chodalirika komanso ubwino wa zotsatira zoyesera.
  Kukonzekera kwa heliXcyto kumaphatikizapo njira ziwiri zofunika: Cyto System Wash, yomwe ingaphatikizidwe mwachindunji mumayendedwe oyesera, ndi Kuyeretsa & Kugona, komwe kumachitidwa ngati chizolowezi chosiyana kuti chisungidwe chidacho chisanayambe kugwiritsidwa ntchito pambuyo pa kuyeza kwautali kwautali usiku wonse kapena osagwiritsidwa ntchito kwa masiku oposa 2.
• Kusamba kwa System ndi Kuyeretsa & Kugona kumafunikira kukhalapo kwa heliX® Maintenance Chip mu tray ya chip.

Kuyeretsa & Kugona Nthawi Zonse

• Njira Yoyeretsa & Kugona imapangidwira kuyeretsa ndi kuzimitsa / kusunga zida. Njirayi imatsuka chubu chamadzimadzi cha chipangizo cha heliX® choyamba ndi madzi a ultrapure kenako ndi 70% ethanol. Pomaliza, machubu onse amatuluka ndi mpweya.
• Njira yoyeretserayi ndi yokhazikika ndipo imatenga pafupifupi mphindi 40. Panthawiyi, ethanol imatulutsidwa mu botolo la madzi, kotero zomwe zili mu botolo ziyenera kutayidwa pambuyo pake.
• Pambuyo pa Kuyeretsa & Kugona, chidacho chimalowa m'malo ogona ndipo sichingagwiritsidwe ntchito mpaka Wake Up & Prime achitidwa. Chip cha heliX® Maintenance ndichofunika pamathamanga onse awiri.
• ZOFUNIKA: Pofuna kupewa kuipitsidwa kwa chubu, onetsetsani kuti mwataya zomwe zili mu botolo lamadzi (madzi / ethanol osakaniza) ndikuchotsa m'chidebe chonyansa pambuyo poyeretsa.

Kutseka ndi kusunga nthawi yaitali

• Kwa nthawi yayitali yosagwiritsidwa ntchito, chonde chotsani botolo lomwe lili ndi madzi ndi ethanol mutatha njira ya Clean & Sleep mu tray ya buffer ndikusiya chubucho chili ndi mpweya.
• Chotsaninso heliX® Maintenance Chip, ndikusunga m'chikwama chake choyambirira. Izi zimalepheretsa kubwereranso ndi kuipitsidwa. Zimitsani chipangizo.

Kusamba kwa cyto system

• heliXcyto System Wash imayeretsa kwambiri microfluidic system pogwiritsa ntchito Cleaning Solution 3 (CS3) mkati mwa mphindi 45. CS3 imatengedwa mu mphindi ziwiritages. Choyamba singano ndi sample loop amadzazidwa ndikumakulitsidwa kuti achotse zoipitsidwa.
• ZINDIKIRANI: Thamangani Cyto System Wash osachepera tsiku ndi tsiku pamene chipangizocho chikugwiritsidwa ntchito. Tikukulimbikitsani kuti muwonjezere njira ya Cyto System Wash pamayendedwe oyeserera mwina pambuyo pa kuyesa kulikonse (pakusintha mzere watsopano wa cell kapena ngati sample quality ndi suboptimal) kapena mpaka kumapeto kwa ntchito yoyeserera. Kusinthana chip yokonza pambuyo pa 50 Cyto System Washes yowonetsedwa ngati Kubadwanso kwatsopano mu tray ya Chip view za Control Chipangizo.

Contact

• Malingaliro a kampani Dynamic Biosensors GmbH
• Perchtinger Str. 8/10
• 81379 Munich
• Germany
• Malingaliro a kampani Bruker Scientific LLC
• 40 Manning Road, Manning Park
• Billerica, MA 01821

USA

• Kuitanitsa Zambiri order.biosensors@bruker.com
• Othandizira ukadaulo support.dbs@bruker.com
• www.dynamic-biosensors.com
• Zida ndi tchipisi amapangidwa ndikupangidwa ku Germany.
• ©2025 Dynamic Biosensors GmbH
• Kugwiritsa Ntchito Kafukufuku Pokha. Osagwiritsidwa ntchito pazachipatala.

FAQ

scIC FAQs

Kodi ndingagwiritsenso ntchito chip cha heliXcyto?

Inde, chip cha heliXcyto ndi chogwiritsidwanso ntchito komanso chotaya. Tsatirani malangizo oyenera oyeretsa ndi kusunga.

Kodi Cholinga cha Chip Maintenance ndi chiyani?

Chip cha Maintenance chimagwiritsidwa ntchito kuyeretsa, kuyesa, ndi kukonza magwiridwe antchito kuti zitsimikizire kuti dongosololi likuyenda bwino.

Kodi ndiyeretse kangati chipangizochi?

The fluidic system ya heliXcyto imakhala yoyera ndi Cyto System Wash ndi Clean & Sleep njira. Dongosolo la microfluidic likulimbikitsidwa kuti liyeretsedwe kudzera pa Cyto System Wash pa Maintenance Chip pambuyo poyesa (makamaka posintha mtundu wa cell) kapena posachedwa kumapeto kwa ntchito yoyeserera. Njira Yoyeretsera & Kugona iyenera kugwiritsidwa ntchito musanagwiritse ntchito mutatha kuyeza kwanthawi yayitali (monga m'mawa mutatha kuyesa usiku wonse) kapena pamene heliXcyto sidzagwiritsidwa ntchito kwa masiku opitilira awiri.

Kodi njira zothetsera RB 1 / madzi / CS 1 / CS 3 / Normalization zitha kusungidwa mpaka liti pazida?

Nthawi zambiri, muyeso uliwonse usanachitike, mayankho onse amawunikidwa pa voliyumu yotsalayo komanso chifukwa cha chipwirikiti kapena mvula. Zikatero yankho liyenera kusinthidwa nthawi yomweyo. Madzi ndi RB 1 azisinthidwa tsiku lililonse ndipo RB 1 imayenera kusefedwa muyeso uliwonse usanachitike. Diluted Normalization solution itha kugwiritsidwa ntchito kwa masiku awiri motsatizana. Kuyeretsa Mayankho amakhala okhazikika kwa masiku atatu.

Kodi ndingagwiritse ntchito chip cha heliXcyto mpaka liti?

Tsiku lotha ntchito la heliXcyto chip likhoza kufufuzidwa mu tabu ya Chip tray mu gawo la Chipangizo mu heliOS. Palibe chitsimikizo cha kukhulupirika kwa chip chomwe chingaperekedwe pambuyo pa tsiku lotha ntchito. Komabe, malinga ngati misampha imakhala yokhazikika, pamwamba pake ndi oyera, ndipo maziko a fulorosenti ali pansi pa odulidwa omwe asonyezedwa mu Chip Test, chip chimatengedwa kuti chingagwiritsidwe ntchito poyeza. Kuyeretsa kunja kwa chip nthawi zonse (Chip Cleaning Kit CCK-1-1 ikulimbikitsidwa kuti ichitike mukagwiritsa ntchito chip kuti mutalikitse moyo wa chip kwambiri.

Kodi ndingagwiritsire ntchito chipangizochi kwa nthawi yayitali bwanji?

Chip cha Maintenance chikufunika kuti chisinthidwe pambuyo pa kutsuka kwa 50 System (kuwerengedwa ngati kusinthidwanso pa Chip tray tabu ya Chipangizo chowongolera mu heliOS).

Kodi HeliXcyto Chip Test iyenera kuchitidwa kangati?

Chip Test imasonkhanitsa zambiri zakumbuyo kwa fluorescence, ukhondo, ndi kukhulupirika kwa chip musanayambe kuyesa kwatsopano. Iyenera kuchitidwa kamodzi kokha pamene chip chatsopano chikugwiritsidwa ntchito, koma tikulimbikitsidwa kuti chichitike kale kapena kumayambiriro kwa kuyesa kulikonse kuti muwone momwe chip chilili. Izi zimathandiza kuti miyeso yoyezera isinthidwe ndi zolakwika za ma sensorgram kuti zitsatidwe ku chip kapena chipangizo.

Kodi chemistry ya analyte ndi chiyani?

Utoto wofiyira kapena wobiriwira wa fulorosenti, motsatana, umaphatikizidwa ndi zida zathu zolembera kudzera pa NHS-esters kupita ku ma amines oyambira pama protein analyte (monga lysines kapena N-terminus). Tsatanetsatane ndi gawo lothetsera mavuto lingapezeke mu heliXcyto Labeling Kit utoto wofiira (Labeling Kit utoto wofiira 2 CY-LK-R2-1) ndi heliXcyto Labeling Kit utoto wobiriwira (Kulemba Kit utoto wobiriwira CY-LK-G1-1 ) zolemba zathu webshopu.

Kodi mungapeze kuti zidziwitso za heliOS ndi chidziwitso cha layisensi?

Njira yolowera zidziwitso ndi zidziwitso za laisensi ikufotokozedwa mu gawo la Kuyika kwa heliOS mu kalozera wa heliXcyto kuchokera kwathu. webtsamba (heliXcyto kalozera). Zambiri zamalayisensi anu ziyenera kusungidwa mufoda ya scIC pa heliXcyto PC Desktop, yomwe Katswiri wathu wa Ntchito adapanga pophunzitsa.

Kodi chisangalalo/kutulutsa mpweya kwa heliXcyto ndi chiyani?

Kusangalatsa kofiira ndi 605-625 nm, kutulutsa (kuzindikira) ndi 655-685 nm. Kusangalatsa kobiriwira 490-510 nm, kutulutsa kobiriwira ndi 525-575 nm.

Ndi kangati komwe ndiyenera kuyeza kuyesa komweku kuti ndidalilikike?

Kuti mukwaniritse ma kinetic odalirika, kubwereza kwa 3-4 kwa miyeso ya 3-concentration Kinetics akulimbikitsidwa kuti azichita m'maselo amtundu umodzi. M'magawo osakanikirana a deta, mayanjano ndi kugawanika kwa zobwereza ziyenera kukhala mkati mwawindo la 2-4.

Kodi kukhudzika kwa miyeso ya scIC ndi chiyani?

Kutsika kwapang'onopang'ono potengera zomwe mukufuna kutsata kumadalira zolemba za analyte, koma nthawi zambiri scIC imatha kuyeza kale ma kinetics pa mamolekyu ochepa a 1000-10000 pa selo. Kuti muwonjezere kukhudzika, ma cell osachepera 5 akulimbikitsidwa kuti alandidwe, ndipo kusanthula kwa DOL ya 5-10 ndi mphamvu ya LED akuyenera kulinganizidwa kuti akwaniritse kuchuluka kwa fluorescence.

Kodi malire ozindikira a heliXcyto ndi otani pamitengo ya kinetic?

Chonde tchulani Mafotokozedwe aukadaulo a heliXcyto kuti mupeze malire aukadaulo aposachedwa, omwe angapezeke mu kalozera wa heliXcyto (kalozera wa heliXcyto). Disociation constant Kd: 10 pM mpaka 1 mM Association mlingo nthawi zonse kon: 1E3 mpaka 1E7 M-1s-1 Dissociation rate nthawi zonse koff: 1E-6 mpaka 1 s-1 Kuti mudziwe zambiri chonde onani kalozera wathu wa heliXcyto kuchokera ku webmalo.

Zolemba / Zothandizira

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Maumboni

• Buku Logwiritsa Ntchito