1. Ukuba ndifumana ityhubhu yeeseli ezikhenkcezisiweyo, ndingayifaka ngqo kwinitrogen engamanzi ukuze igcinwe?
Kwiimeko ezininzi, iiseli ezithuthwa kumkhenkce owomileyo (-80°C) zinokubuyiselwa kwinitrogen engamanzi emva koko inyibilikiswe ngokukhawuleza emva koko.Nangona kunjalo, ukusebenza kweeseli kunokuncitshiswa emva konyango olunjalo.Kweminye imigca yeeseli ebuthathaka, oku kunokwenza ukubuyiswa kweeseli kube nzima ngakumbi.Le nto icingelwa ukuba ibangelwa yinguqu kwisakhiwo seekristale zeqhwa ngaphakathi kweeseli ngenxa yokutshintsha kweqondo lokushisa.Ngoko ke kuyacetyiswa ukuba iiseli kufuneka zinyibilikiswe kwaye zikhuliswe ngokukhawuleza emva kokuba zifunyenwe.Nciphisa ixesha lokugcina kwi -80°C.Eli qondo lobushushu lisetyenziselwa ukuthutha kuphela.
2. Ngawaphi amanyathelo okhuseleko ekufuneka athatyathwe xa kususwa iiseli kwinitrogen engamanzi ukuze zibuyiselwe?
I-Cryotubes yeseli kwinitrogen engamanzi engatywinwanga ngokupheleleyo kwaye inenitrogen engamanzi evuzayo kuzo inokubangela ugqabhuko-dubulo ukuba ubushushu be-cryotube bunyuka ngokubukhali xa inyibilika.Ngoko ke kucetyiswa ukuba iiglavu kunye neeglavu zokukhusela zinxitywe xa kususwa iiseli kwi-nitrogen engamanzi.Ukuvuselela, ityhubhu yokukhenkceza kufuneka ishukunyiswe ngokuqhubekayo kwindawo yokuhlambela amanzi angama-37 ° C ukuze inyibilike ngokupheleleyo isisombululo sokukhenkceza ngaphakathi kwemizuzu eyi-1-2.Emva koko, sula umphandle wetyhubhu nge-alcohol yosula, emva koko uyifake kwitafile ecocekileyo kakhulu kwaye udlulisele iiseli kwityhubhu ye-centrifuge ene-10 ml yenkcubeko yenkcubeko eyongeziweyo, i-centrifuge kwi-1000 rpm imizuzu emi-5-10, lahla amandla amakhulu, yongeza inani elifanelekileyo lenkcubeko yenkcubeko kwaye utofe iflask yenkcubeko kwaye ufukamele kwi-5% CO2 incubator.
3. Kutheni le nto kufuneka iiseli zigcinwe kwisigaba somphunga setanki yenitrogen engamanzi kunakwisigaba solwelo?
Iiseli ezigcinwe kwisigaba segesi senitrogen engamanzi kunokwenzeka ukuba zivuselelwe.Ngelixa kwisigaba solwelo senitrogen yolwelo, ukuba iityhubhu zelyophilisation azivalwanga kakuhle okanye zinokuvuza, ukudibana ngqo phakathi kweeseli kunye nenitrogen engamanzi kunokuphazamisa ukusebenza kweeseli emva kokunyibilika.
4. Kwiiseli ezixhonyiweyo, ndingayitshintsha njani indlela yenkcubeko?
Ukuhlakulela iiseli zokunqunyanyiswa kungenziwa ngokudibanisa nje i-fresh medium (ukuba indawo ivumela) okanye ngokwahlula iiseli ukusuka kwi-medium yakudala nge-centrifugation (100 xg yemizuzu emi-5) kwaye emva koko kumiswe kwakhona iiseli zemvula kwindawo entsha.Nangona kunjalo, kwimigca yeeseli ezininzi ezimisiweyo, ukongeza nje okuphakathi yeyona ndlela ingcono.Nokuba yeyiphi na indlela, okuphakathi kufuneka kuhlaziywe phambi kokuba iiseli zifikelele kuxinano lwazo olukhulu lokugcwala.Ubuninzi be-saturation yeeseli buhluka phakathi kwe-3 x 10 5 kunye ne-2 x 10 6 kuxhomekeke kumgca weseli kunye neemeko zenkcubeko (ukuphumla okanye ukuvuselela, amanqanaba oksijini, njl.).Iiseli kufuneka zihlanjululwe kugxininiso lweeseli ezisezantsi ukuvumela ukubuyiswa ngokwaneleyo kwezondlo ukugcina iiseli zikhula ngokwe-logarithmically.Ukuba i-medium iguqulwa nje ngaphandle kokunciphisa ukuxinana kweeseli, iiseli ziya kuphelisa ngokukhawuleza okuphakathi kwaye zife.Ukuba iiseli zihlanjululwe ngaphantsi koxinano lwazo oluncinci, ziya kungena kwinqanaba le-lag kwaye zikhule ngokucothayo okanye ziya kufa.Umgca weseli omisiweyo ngamnye unoxinano olwahlukileyo lokuxinana kunye nesithuba sokudlula, ngoko ke ukubalwa kweeseli zemihla ngemihla yindlela yokubeka iliso kwimigca yeseli emisiweyo*.
5. Leliphi inqanaba le-CO2 elicetyiswayo kwinkcubeko yeeseli?
Nangona amanqanaba e-CO2 kwiinkqubo zenkcubeko yeeseli ukusuka kwi-0.03% ukuya kwi-40% (ngokuqhelekileyo malunga ne-0.03% ye-CO2 emoyeni), kuxhaphake kakhulu ukuba akukho CO2 eyongeziweyo emoyeni okanye i-CO2 yoxinaniso lwe-5% ukuya kwi-10%.Kubalulekile ukulungelelanisa i-concentration ye-sodium bicarbonate phakathi ukulinganisela kunye nenqanaba le-CO2 kwisigaba segesi.Iiseli zivelisa i-CO2 kwaye zifuna inani elincinci le-carbonic acid yokukhula kunye nokuphila.Ukuba akukho CO2 eyongeziweyo kwaye iiseli ziyaphindaphindeka, i-4 mM (0.34 g/L) ye-anhydrous sodium bicarbonate ingasetyenziswa.Nangona kunjalo, isiciko seflask yenkcubeko kufuneka siqiniswe ngeli xesha.Ukuba inkqubo yenkcubeko idinga i-5% okanye i-10% ye-CO2, sebenzisa i-23.5 mM (1.97 g / L) okanye i-47 mM (3.95 g / L) ye-sodium bicarbonate kwi-37 ° C, ngokulandelanayo, kunye ne-pH yokuqala malunga ne-7.6.Ngaphantsi kwezi meko, i-flask kufuneka ishiywe ingafakwanga okanye isitya sePetri kufuneka sisetyenziswe ukugcina ukulingana kwegesi.
6. Kutheni ezinye iiseli zifuna i-sodium pyruvate?Ingakanani i-pyruvate ye-sodium endiyifake kwi-medium?
I-Pyruvate yi-organic acid metabolite kwindlela ye-glycolytic * engena lula kwaye ishiya iseli.Ngoko ke, ukongezwa kwe-sodium pyruvate ukuya kumbindi kunika zombini umthombo wamandla kunye nomthombo wekhabhoni ye-anabolism, inceda ukugcina iiseli ezithile ezithile, inceda nge-cloning yeseli okanye iyadingeka xa ukugxilwa kwe-serum phakathi kuyancipha.I-sodium pyruvate inceda ekunciphiseni i-cytotoxicity eyenziwe yi-fluorescence.I-sodium pyruvate idla ngokudibaniswa kwinqanaba lokugqibela le-1 mM.Izisombululo ze-sodium pyruvate ezithengiswayo zihlala ziyi-100 mM isisombululo sokugcina (100X).
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Ixesha lokuposa: Jun-21-2022