Qhov Tseem Ceeb Qhov txawv - Sov vs Cold Trypsinization
Sov thiab Txias Trypsinization yog ob txoj hauv kev siv hauv enzymatic disaggregation ntawm cov hlwb hauv cov tsiaj cell culturing. Qhov sib txawv tseem ceeb ntawm qhov sov thiab txias trypsinization, raws li cov npe qhia, nyob ntawm qhov kub ntawm qhov trypsin ntxiv rau kev sib cais ntawm tes. Ua kom sov trypsinization tshwm sim nyob rau hauv qhov kub thiab txias (36.5 - 37 0 C) whereas txias trypsinization tshwm sim nyob rau hauv qis-kub.
Thaum lub sijhawm txheej txheem ntawm kev cog qoob loo ntawm cov tsiaj txhu, muaj peb txoj hauv kev tseem ceeb tau siv thiab tau ua pov thawj kom ua tiav. Peb txoj kev suav nrog kev sib cais ntawm cov hlwb, enzymatic disaggregation ntawm cov hlwb thiab cov txheej txheem kev cog qoob loo. Lub enzymatic disaggregation ntawm cov hlwb ua rau kev sib cais ntawm cov hlwb thiab nws yog ua los ntawm cov protein-degrading enzyme trypsin. Yog li, cov txheej txheem no hu ua Trypsinization. Trypsinization tuaj yeem ua tiav nyob rau hauv ob qhov sib txawv xws li sov Trypsinization thiab Cold Trypsinization. Ua kom sov trypsinization yog txoj kev kho cov hlwb nrog trypsin nyob rau hauv qhov chaw sov ntawm qhov kub ntawm 36.5 - 37 0 C. Txias trypsinization yog cov txheej txheem ntawm trypsin kev kho mob uas tshwm sim nyob rau hauv txias txias dua nyob rau hauv cov dej khov uas tsis tshua muaj kub.
Sov Trypsinization yog dab tsi?
Trypsinization tuaj yeem ua tiav txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau cais cov hlwb los tsim cov kab lis kev cai ntawm tes. Trypsin yog ib qho protein degradation enzyme, thiab cov enzyme sib xyaw siv hauv trypsinization tuaj yeem yog cov khoom siv raw los yog cov khoom ua kom huv. Cov kua txiv hmab txiv ntoo tau hais tias ua tau zoo dua hauv cov protein lysis thiab cell disintegration vim nws muaj lwm cov enzymes degradative.
Sov trypsinization yog qhov feem ntau siv enzymatic txoj kev rau cell disaggregation uas tshwm sim nyob rau hauv lub kub kub. Ua ntej kev kho mob los ntawm trypsinization, cov ntaub so ntswg xav tau raug txiav mus rau hauv me me pieces. Nws pab txoj kev yooj yim disaggregation. Cov ntaub so ntswg raug ntxuav hauv ib qho tshwj xeeb tshaj tawm hu ua Dissection Basal S alt nruab nrab.
Thaum ua tiav cov kauj ruam ntxuav, cov hlwb hloov mus rau hauv lub raj mis uas muaj cov enzyme nquag, uas yog trypsin. Raws li cov txheej txheem no qhia txog kev ua kom sov trypsinization raws tu qauv, cov trypsin tau muab tso rau ntawm qhov kub ntawm ib ncig ntawm 37 0 C rau li plaub teev.
Daim duab 01: Trypsin
Cov ntsiab lus sib xyaw thiab ua rau ntxhov siab los ntawm kev siv cov txheej txheem centrifugation kom yooj yim ntawm cov txheej txheem thiab kom ceev cov txheej txheem disaggregation. Thaum lub sijhawm pom zoo ua tiav, cov hlwb tuaj yeem muab tau los ntawm cov supernatant. Cov hlwb muab tau los ntawm cov supernatant ces incubated ntawm qhov kub thiab lub sij hawm.
Cold Trypsinization yog dab tsi?
Cold trypsinization yog lwm hom trypsinization uas tshwm sim nyob rau hauv txias txias. Hauv cov txheej txheem no, cov hlwb uas tau txiav thiab ntxuav yog muab tso rau hauv vials ntawm cov dej khov thiab tom qab ntawd soaked nrog trypsin. Lub sij hawm ntub dej ntau dua - txog 6 - 24 teev.
Tom qab ua tiav cov txheej txheem soaking, cov trypsin raug tshem tawm ntawm cov cell lysate, thiab cov ntaub so ntswg yog ntxiv incubated ntawm 370 C rau li 20 - 30 feeb. Lub disaggregation ntawm cov hlwb yog coj los ntawm rov pipetting ntawm cov ntaub so ntswg sib tov. Qhov no yuav tso cai rau cov hlwb kom dissociate ntawm daim nyias nyias thiab tuaj rau lub supernatant. Thaum cov hlwb nyob rau hauv supernatant, lawv tau incubated thiab loj hlob ntawm qhov xav tau kub thiab lub sij hawm.
Txoj kev sim ua kom txias muaj ntau yam zoo
- Cov txiaj ntsig ntau dua ntawm cov hlwb siv tau raws li kev puas tsuaj ntawm tes raug txo qis. Kev puas tsuaj ntawm tes yog txo qis los ntawm kev tsis siv cov kauj ruam centrifugation.
- txoj kev yooj yim heev.
- Less laborious.
Qhov kev txwv tseem ceeb ntawm txoj kev ua kom txias txias yog qhov ntau tsis tuaj yeem siv hauv ib qho piv txwv.
Dab tsi zoo sib xws ntawm sov thiab txias Trypsinization?
- Ob chav sov thiab txias Trypsinization siv cov enzyme trypsin rau kev sib cais ntawm cov hlwb.
- Ob qho tib si sov thiab txias Trypsinization txheej txheem yog siv nyob rau hauv cell kab lis kev cai txheej txheem rau disaggregation ntawm hlwb.
- Nyob rau hauv ob qho tib si sov thiab txias Trypsinization cov txheej txheem, cov hlwb tau muab los ntawm supernatant.
Qhov txawv ntawm qhov sov thiab txias trypsinization yog dab tsi?
Sov vs Cold Trypsinization |
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| Sov trypsinization yog txoj kev kho cov hlwb nrog trypsin nyob rau hauv qhov chaw sov ntawm qhov kub ntawm 36.5 - 37 0. | Cold trypsinization yog txheej txheem ntawm trypsin kev kho mob uas tshwm sim nyob rau hauv txias txias dua nyob rau hauv cov dej khov uas tsis tshua muaj kub. |
| Protocol | |
| Cov ntaub so ntswg txiav tau khaws cia ntawm 37 0 C txuas ntxiv mus thoob plaws hauv txheej txheem. | Cov nqaij ntuag yog thawj zaug khaws cia ntawm cov dej khov txias thiab tom qab ntawd khaws cia ntawm 37 0. |
| Sov trypsinization tshwm sim ntawm 36.5 - 37 0 | Cold trypsinization tshwm sim ntawm cov dej khov txias. |
| Lub sijhawm tsawg dua yuav tsum tau ua rau tag nrho cov txheej txheem (kwv yees li 4 teev) ntawm qhov sov so trypsinization. | Lub sijhawm ntev dua (kwv yees li 6 - 24 teev) rau kev ua kom txias txias. |
| Yield of Viable Cells | |
| Tsawg hauv sov trypsinization. | siab txias trypsinization. |
| Kev siv centrifugation | |
| Centrifugation yuav tsum tau rau kev sib cais ntawm cov hlwb hauv sov trypsinization. | Centrifugation tsis tas yuav tsum muaj nyob rau hauv txias trypsinization. |
| Cov ntaub so ntswg pib ntau rau Trypsinization | |
| Ntau ntau ntawm cov ntaub so ntswg tuaj yeem siv rau hauv sov trypsinization. | Ib qho me me ntawm cov ntaub so ntswg tuaj yeem siv rau hauv qhov txias trypsinization. |
| Cell Damage | |
| High vim yog centrifugation hauv sov trypsinization. | Tsawg dua vim txias trypsinization. |
Summary – Sov vs Cold Trypsinization
Trypsinization yog txoj hauv kev siv cov protein-degrading enzyme trypsin rau kev sib cais thiab kev npaj ntawm cov kab lis kev cai hauv lub cev thaum lub sij hawm cellculturing txheej txheem. Muaj ob txoj hauv kev tseem ceeb ntawm trypsinization raws li qhov kub thiab txias siv thaum lub sijhawm ua haujlwm. Lawv sov thiab txias trypsinization. Ua kom sov trypsinization yog ua tiav ntawm 37 0 C whereas txias trypsinization yog ua nyob rau hauv cov dej khov-txias. Txawm hais tias txias trypsinization yuav siv sij hawm ntev dua rau kev ua tiav, nws tau hais tias muaj cov txiaj ntsig zoo dua ntawm cov hlwb. Qhov no yog vim qhov tseeb tias kev puas tsuaj ntawm tes raug txo qis hauv qhov txias trypsinization vim nws tsis siv cov kauj ruam centrifugation. Qhov no yog qhov txawv ntawm qhov sov thiab txias trypsinization.
