Iingcebiso ezisisiseko zokuthunga zenkcubeko

Iingcebiso ezisisiseko zokuthunga zenkcubeko

Iiseli ziqhubeka nemetabolism yazo ngexesha lokukhula kwesiqhelo kukukhula, ezifuna inxaxheba kwiiproteases ezahlukeneyo. Xa iqondo lobushushu liphosa ngaphantsi -70 ° C, ukuyeka ukusebenza. Ke ngoko, kwindalo enamaqondo obushushu asezantsi, iiseli zinokuyeka imisebenzi yazo ye-metabolic kwaye ifake imeko engapheliyo evumela ukugcinwa kwexesha elide.

1. Izinto zovavanyo

Isiseko sezinto:

Inkcubeko esisiseko

I-serum (i-FBS eqhelekileyo / i-NBS)

Umsebenzi wokugqibela

I-Steile Dimyethyl SulfoXide (DMSO)

I-Stead PBs phosphate buffer i-buffer i-stor

Umpu wepayi

Umpu wombane

Ukulungiswa kovavanyo

A) Ukulungiswa kwesisombululo sokuqaqanjelwa:

Iiseli ngokubanzi: I-55% ephakathi ye-Bassium + i-40% ye-BOVIne SUUM (FBS / NBS) + 5% DMO

Iiseli ezibalulekileyo: I-90% ye-Bovine serum (FBS / NBS) + i-10% DMO

I-alquot isisombululo esilungiselelwe i-stooprest kwi-15 ml Centrifuge Tube [indlwane] kwaye igcine kwi-4 ° C ukuze isetyenziswe kamva.

(b) Ukulungiselela iiseli ukuba zibe ngumkhenkce:

Phambi kokuba ukhulule iiseli, khetha iiseli ezinesilinganiselo sokukhula okuhle kunye nenqanaba lokukhula kwe-logo kwaye libeke endaweni yeeyure ezili-12 ukuya kwezi-24 ngaphambi kokukhulula ukugcina imeko.

2. Inkqubo yovavanyo

1. Qwalasela uxinzelelo lweeseli ukuba libe liqhwa, elinokuba ngama-80% ~ 90%. Sebenzisa umpu wepayiphu ukuze uvuselele i-Windourium endala, yongeza ii-PB ezi-stele ukuhlamba iiseli ezi-1-2, kwaye ususe iphakathi eseleyo kwinkcubeko.

2. Yongeza isixa esifanelekileyo se-trypsin okanye ijusi yokugaya ukuvumela i-matpsin ukuba ingene kwiiseli, kwaye ibeke kwi-incubator ukuze igaye. Jonga imeko yeeseli eziphantsi kwe-microscope: I-cytoplasm ibuyisa kwaye iiseli azisasebenzi emakhishini. Okwangoku, yongeza isisombululo sokumisa ukumisa inkqubo yokugaya.

3. I-bubble ngobunono iiseli ezinencam ukwenza ukumiswa kweseli ukwenza ukumiswa kweseli kunye ne-Centrifuyisi yokumiswa kweseli kwi-1000 RPM imizuzu emi-3-5.

Lahla i-supernatant, yongeza isixa esifanelekileyo sesisombululo se-frezing, kunye nomphunga wokwenza ubumdaka ukwenza iiseli zibala ngokufanayo. Lungisa ukungangqinelani kweseli ngokufumana i-crypresment ukwenza i-5 × 106 / ml ~ ~ 17 / ml.

4. Sebenzisa umpu wepayitha ukwahlula iityhubhu ze-crygenic ngokwesikhundla esilindelekileyo, kwaye usebenzise umatshini we-cappic ozenzekelayo uye kwi-cap kunye netywina.

5. Inkqubo yokuSetyenziswa kwe-Crytoopre yokuSetyenziswa kweMigangatho yokuphola ye--1 ° C /2 ye-C /2, kwaye iiseli ze-CREALD zinokuqengqeleka ngokuthe ngcembe ngokwamanqanaba alandelayo: iqondo lobushushu igumbi → i-4 (i-20min) → I-20 ° C (30min) → I-30min) → -80 ° C (ngobusuku bonke) → ukugcinwa kwexesha elide kwi-nitrogen yolwelo.

Ingagcinwa ngokuthe ngqo kwi-freezer kwi-30 ° CR ubusuku kwaye idluliselwe kwi-nitrogen yolwelo ukuze igcinwe.

I-Youboour yezonyango i-proterise ye-tech-tech kwi-R & D, imveliso kunye nentengiso ye-biomeds enokuthengiswa. Iingcebiso zepayiphu, imibhobho, iplate ye-PCR, iimveliso zenkcubeko kunye nezinye izinto zelebhu yenyama. I-YongYyee yezonyango ifanele ithembekile yakho kunye nokhetho! Wamkelekile ukunxibelelana!