請(qǐng)問(wèn)：實(shí)驗(yàn)中我該如何選用合適的培養(yǎng)基？
發(fā)布時(shí)間：
2022-12-24
作者：
一次性培養(yǎng)基生產(chǎn)廠家
培養(yǎng)某一類(lèi)型細(xì)胞沒(méi)有固定的培養(yǎng)條件。在MEM中培養(yǎng)的細(xì)胞，很可能在DMEM或M199中同樣很容易生長(zhǎng)?？傊?，選MEM做粘附細(xì)胞培養(yǎng)，RPMI-1640做懸浮細(xì)胞培養(yǎng)，各種目的無(wú)血清培養(yǎng)的選是AIMV培養(yǎng)基[SFM]。
2.為什么要熱滅活血清？
加熱可以滅活補(bǔ)體系統(tǒng)。激活的補(bǔ)體參與溶解細(xì)胞事件，刺激平滑肌收縮，細(xì)胞和血小板釋放組胺，激活淋巴細(xì)胞和巨噬細(xì)胞。在進(jìn)行免疫學(xué)研究，培養(yǎng)ES細(xì)胞，昆蟲(chóng)細(xì)胞和平滑肌細(xì)胞時(shí)，推薦使用熱滅活血清。
3.L-谷氨酰胺在細(xì)胞培養(yǎng)中重要嗎？它在溶液中不穩(wěn)定嗎？
L-谷氨酰胺在細(xì)胞培養(yǎng)時(shí)是重要的。脫掉氨基后，L-谷氨酰胺可作為培養(yǎng)細(xì)胞的能量來(lái)源，參與蛋白質(zhì)的合成和核酸代謝。L-谷氨酰胺在溶液中經(jīng)過(guò)一段時(shí)間后會(huì)降解，但是確切的降解率一直沒(méi)有最終確定。L-谷氨酰胺的降解導(dǎo)致氨的形成，而氨對(duì)于一些細(xì)胞具有毒性。
4.GlutaMAX-I是什么？培養(yǎng)細(xì)胞如何利用GlutaMAX-I？這個(gè)二肽有多穩(wěn)定？
GlutaMAX-I二肽是L-谷氨酰胺的衍生物，將其不穩(wěn)定的α-氨基用L-丙氨酸來(lái)保護(hù)。一種肽酶逐漸裂解二肽，釋放L-谷氨酰胺供利用。
GlutaMAX-I二肽非常穩(wěn)定，即使在121磅滅菌20分鐘，GlutaMAX-I二肽溶液有最小的降解，如果在相同條件下，L-谷氨酰胺幾乎完全降解。
5.為什么培養(yǎng)基中可以省去加酚紅？
酚紅在培養(yǎng)基中被用來(lái)作為PH值的指示劑：中性時(shí)為紅色，酸性時(shí)為黃色，堿性時(shí)為紫色。研究表明，酚紅可以模擬固醇類(lèi)激素的作用[特別是雌激素]。為避免固醇類(lèi)反應(yīng)，培養(yǎng)細(xì)胞，尤其是哺乳類(lèi)細(xì)胞時(shí)，用不加酚紅的培養(yǎng)基。由于酚紅干擾檢測(cè)，一些研究人員在做流式細(xì)胞檢測(cè)時(shí)，不使用加有酚紅的培養(yǎng)基。
6.如何用臺(tái)盼蘭計(jì)數(shù)活細(xì)胞？
用無(wú)血清培養(yǎng)基把細(xì)胞懸液稀釋到200-2000個(gè)/毫升，在0.1毫升的細(xì)胞中加入0.1毫升的0.4％的臺(tái)盼蘭溶液。輕輕混勻，數(shù)分鐘后，用血球計(jì)數(shù)板計(jì)數(shù)細(xì)胞。活細(xì)胞排斥臺(tái)盼蘭，因而染成藍(lán)色的細(xì)胞是死細(xì)胞。
7.如何消除組織培養(yǎng)的污染？
當(dāng)重要的培養(yǎng)污染時(shí)，研究者可能試圖消除或控制污染。首先，確定污染物是細(xì)菌，真菌，支原體或酵母，把污染細(xì)胞與其它細(xì)胞系隔離開(kāi)，用實(shí)驗(yàn)室消毒劑消毒培養(yǎng)器皿和超凈臺(tái)，檢查HEPA過(guò)濾器。
高濃度的抗生素和抗霉菌素可能對(duì)一些細(xì)胞系有毒性，因而，做劑量反應(yīng)實(shí)驗(yàn)確定抗生素和抗霉菌素產(chǎn)生毒性的劑量水平。這點(diǎn)在使用抗生素如兩性霉素B和抗霉菌素如泰樂(lè)菌素時(shí)尤其重要。下面是推薦的確定毒性水平和消除培養(yǎng)污染的實(shí)驗(yàn)步驟。
1)在無(wú)抗生素的培養(yǎng)基中消化，計(jì)數(shù)和稀釋細(xì)胞，稀釋到常規(guī)細(xì)胞傳代的濃度。
2)分散細(xì)胞懸液到多孔培養(yǎng)板中，或幾個(gè)小培養(yǎng)瓶中。在一個(gè)濃度梯度范圍內(nèi)，把選擇抗生素加入到每一個(gè)孔中。例如，兩性霉素B推薦下列濃度，0.25，0.50，1.0，2.0，4.0,8.0mg/ml。
3)每天觀測(cè)細(xì)胞毒性指標(biāo)，如脫落，出現(xiàn)空泡，匯合度下降和變圓。
4)確定抗生素毒性水平后，使用低于毒性濃度2-3倍濃度的抗生素的培養(yǎng)液培養(yǎng)細(xì)胞2-3代。
5)在無(wú)抗生素的培養(yǎng)基中培養(yǎng)細(xì)胞一代。
6)重復(fù)步驟4。
7)在無(wú)抗生素的培養(yǎng)基中培養(yǎng)4-6代，確定污染是否以已被消除。
8.培養(yǎng)基中丙酮酸鈉的作用是什么？
丙酮酸鈉可以作為細(xì)胞培養(yǎng)中的替代碳源。盡管細(xì)胞更傾向于以葡萄糖作為碳源，但是，如果沒(méi)有葡萄糖的話，細(xì)胞也可以代謝丙酮酸鈉。
9.Hank’s平衡鹽溶液[HBS]要在空氣中使用，不需要CO2培養(yǎng)箱。原因是什么？Hank’s平衡鹽溶液[HBS]和Earle’s平衡鹽溶液[EBS]有什么本質(zhì)的功能差別？
HBS和EBS的主要差別在于碳酸氫鈉的水平，碳酸氫鈉的含量在Eagles(2.2g/L)中比在Hanks(0.35g/L)中高。碳酸氫鈉需用高水平的CO2平衡，以維持溶液的PH值。Eagles液在空氣水平的CO2中，溶液會(huì)變堿，Hanks液在CO2培養(yǎng)箱中會(huì)變酸。如果希望在CO2培養(yǎng)箱中保存組織，需要用Eagles液。如果僅僅是清洗將要在細(xì)胞培養(yǎng)基中儲(chǔ)存的組織，用Hanks液就可以了。
10.Qualified和Certified胎牛血清有什么差別?
Certified胎牛血清包括了Qualified胎牛血清執(zhí)行的所有的標(biāo)準(zhǔn)檢驗(yàn)程序，而且除了這些標(biāo)準(zhǔn)檢測(cè)，Certified胎牛血清還有如下一些附加的檢測(cè)：
End-PointDeterminationofEndotoxinContent
噬菌體檢驗(yàn)
生物化學(xué)檢測(cè)
激素的檢測(cè)
血紅蛋白檢測(cè)
Sf9細(xì)胞生長(zhǎng)促進(jìn)及方法學(xué)檢測(cè)
11.二價(jià)離子抑制胰蛋白酶活性嗎？使用胰蛋白酶時(shí)加入EDTA的目的是什么？
二價(jià)離子的確抑制胰蛋白酶活性。EDTA用來(lái)螯合游離的鎂離子和鈣離子，以便保持抑制胰蛋白酶的活性。建議胰蛋白酶處理細(xì)胞前，用EDTA清洗細(xì)胞，以消除來(lái)自培養(yǎng)基中所有的二價(jià)離子。
12.制備lipid-DNA的方法會(huì)影響轉(zhuǎn)染效率嗎？
是的。對(duì)于LIPOFECTAMlNEReagent，稀釋試劑在100μlOPTI-MEM中，稀釋DNA在100μlOPTI-MEM中?；旌蟽煞N溶液在室溫下孵育15分鐘。對(duì)于LIPOFECTINReagent，在加入DNA溶液前允許稀釋的試劑和培養(yǎng)基孵育至少30分鐘可以增加3倍的效率。確保復(fù)合物在沒(méi)有血清的情況下形成。孵育15分鐘后，在復(fù)合物中加入含有血清的培養(yǎng)基[800μl]。[注意以上是35mm培養(yǎng)皿使用體積]。對(duì)于LIPOFECTAMlNEPlusReagentDNA應(yīng)該在與脂質(zhì)體混合之前首先與PlusReagent混合。LIPOFECTAMlNE2000的操作步驟允許在一個(gè)很小的體積下混合DNA和脂質(zhì)體，接下來(lái)可以不需要換培養(yǎng)基的情況下直接加入。
13.我使用SF900Ⅱ時(shí)，細(xì)胞生長(zhǎng)良好，但是為什么我的蛋白產(chǎn)物不如使用Graces液和10%胎牛血清時(shí)效果好？
如果目的蛋白是一個(gè)后期蛋白，它將與蛋白酶一起表達(dá)，這些蛋白酶將會(huì)作用于目的蛋白。在加有血清的培養(yǎng)基中，這些蛋白酶將作用到血清中的蛋白，從而使目的蛋白產(chǎn)品保持完好。在無(wú)血清配方中，蛋白酶作用的唯一底物是你的目的蛋白。為了避免這一問(wèn)題，加入一些蛋白酶抑制劑或加入少量的血清[少于1％]，讓血清給蛋白酶提供作用底物。
14.如何檢測(cè)內(nèi)毒素(熱源)水平？
LAL[LimulusAmebocyteLysate]試驗(yàn)是可用的最敏感和特異的檢測(cè)細(xì)菌內(nèi)毒素的方法。Levin和Bang發(fā)現(xiàn)細(xì)菌可引起鱟[Limuluspolyphemus]血管內(nèi)的凝集作用。內(nèi)毒素啟動(dòng)一個(gè)細(xì)胞內(nèi)酶原系統(tǒng)[絲氨酸蛋白酶級(jí)聯(lián)反應(yīng)系統(tǒng)]，通過(guò)修飾凝集素，產(chǎn)生一種透明的膠，LAL中的凝固蛋白，從而，形成不可溶的基質(zhì)。LAL試劑緩沖的鱟[血細(xì)胞]裂解物。
胎牛血清的內(nèi)毒素檢測(cè)是在GrandIsland，按照手冊(cè)上Gel-clot方法進(jìn)行的。在對(duì)血清產(chǎn)品進(jìn)行內(nèi)毒素檢測(cè)前，用無(wú)熱源的水1：10稀釋樣品。稀釋樣品沸水育5分鐘，以消除抑制劑。[通常，血液中的內(nèi)毒素結(jié)合成份的出現(xiàn)會(huì)抑制凝膠過(guò)程，使內(nèi)毒素不能與LAL反應(yīng)。樣品預(yù)先熱處理可以消除這種抑制作用。]
15.我可以使用固體形式的MurashigeSkog培養(yǎng)基嗎？
如果使用固體形式的培養(yǎng)基，需要加入瓊脂。瓊脂加入前要先滅菌。應(yīng)該避免MS培養(yǎng)基的直接滅菌，應(yīng)該高壓滅菌瓊脂溶液，然后把融化的瓊脂溶液加入到MS培養(yǎng)基中。
16.20℃下配制的緩沖液，在較高或較低的溫度下PH值會(huì)改變嗎？
對(duì)于普通使用的緩沖液，PH值隨溫度變化而變化。
下表列出溫度改變10℃時(shí)，PH值的變化情況
例如20℃下配制PH7.4Tris緩沖液,40℃時(shí)PH值為7.4-[2x0.310]＝6.78
17.室溫下(25℃)配制的Tris-HCl溶液，在37℃使用時(shí)PH值是多少？
緩沖液的PH值隨溫度變化而變化。下表列出了50mMTris-HC溶液在4℃，25℃，37℃時(shí)，不同的PH值。
18.昆蟲(chóng)細(xì)胞培養(yǎng)的最適PH值和滲透壓是多少？
生長(zhǎng)培養(yǎng)基的PH值對(duì)細(xì)胞的增值和病毒或重組蛋白的生產(chǎn)均會(huì)產(chǎn)生影響。對(duì)于大部分鱗翅類(lèi)昆蟲(chóng)細(xì)胞系，在PH值6.0-6.4范圍的大部分應(yīng)用效果良好。培養(yǎng)鱗翅類(lèi)昆蟲(chóng)細(xì)胞系時(shí)，培養(yǎng)基的最適滲透壓是345-380mOsm/kg.。為保證可靠和持久的細(xì)胞培養(yǎng)方式，減少技術(shù)問(wèn)題，保持PH值和滲透壓在以上所列的范圍之內(nèi)。
19.HighFive細(xì)胞有任何其它名稱(chēng)嗎？
HighFive細(xì)胞也被稱(chēng)為T(mén)richoplasiani5B1-4和BTI-TN-5B1-4。
20.在HighFive無(wú)血清培養(yǎng)基中去污劑的濃度是多少？
HighFive無(wú)血清培養(yǎng)基中去污劑的濃度：0.025g/LTween-80，1.0g/LPluronicPoly-all。
21.HighFive細(xì)胞用多大的密度凍存？
3.0x10E6cells/ml
22.在我的果蠅培養(yǎng)基中發(fā)現(xiàn)形成白色沉淀，加熱后溶解。它是什么？對(duì)我的細(xì)胞有害嗎？
可能是谷氨酰胺沉淀，但是更可能是L-酪氨酸沉淀。培養(yǎng)基中谷氨酰胺的濃度比典型的2mM高6倍。酪氨酸的濃度比在RPMI1640中高25倍，而且比谷氨酰胺更加難以溶解。沉淀也可能是不止一種成分的復(fù)合物。它可能是由于貯存在局部溫度較低的地方引起。只要沉淀在培養(yǎng)條件下可以溶解，對(duì)實(shí)驗(yàn)不會(huì)有不利的影響。
23.如何從T25瓶中轉(zhuǎn)移sf9細(xì)胞？能用胰蛋白酶消化嗎？
我們強(qiáng)力推薦使用脫落細(xì)胞的方法，因?yàn)檫@項(xiàng)技術(shù)破壞性最小，生活力最高。通過(guò)使用巴氏德吸液管，讓細(xì)胞上培養(yǎng)基流動(dòng)。作為一種選擇你也可以輕輕拍打培養(yǎng)瓶。只有在絕對(duì)必要的情況下，才使用胰酶消化細(xì)胞。
胰酶消化一個(gè)T25瓶的sf9細(xì)胞：
1)去除培養(yǎng)基。
2)用2ml1xPBS[足以覆蓋細(xì)胞表面]洗滌細(xì)胞，去除PBS。
3)加入2ml1x胰酶EDTA[恰好覆蓋細(xì)胞表面]。
4)37℃孵育5到10分鐘。在儀器下檢測(cè)看到5分鐘后它們正在向上移動(dòng)。
5)向細(xì)胞中加入2ml細(xì)胞培養(yǎng)液，移入錐形管，用2ml培養(yǎng)液洗瓶壁，移入同一錐形管中。[培養(yǎng)基中的FBS終止了胰酶的活性。]
6)離心[1100rpm]沉淀細(xì)胞。去除培養(yǎng)基。
7)用新的培養(yǎng)基重新懸浮細(xì)胞。傳代。
24.在Sf9,Sf21,和highFive細(xì)胞懸浮培養(yǎng)時(shí)，肝素的使用量是多少？
為了防止懸浮培養(yǎng)細(xì)胞聚集的形成，使用肝素濃度為10單位/毫升細(xì)胞懸液。
25.如何評(píng)估ES細(xì)胞合格的胎牛血清？
使用D3ES細(xì)胞。這是一個(gè)對(duì)于胎牛血清中生長(zhǎng)促進(jìn)，生長(zhǎng)抑制和分化因子非常敏感的細(xì)胞系。
相關(guān)生長(zhǎng)效率分析：
當(dāng)ES細(xì)胞以非常低的密度傳入包含10％胎牛血清的生長(zhǎng)培養(yǎng)基中，檢測(cè)開(kāi)始和支持ES細(xì)胞克隆的能力。
細(xì)胞毒分析：
當(dāng)以非常低的密度傳入包含30％胎牛血清的生長(zhǎng)培養(yǎng)基中，檢測(cè)ES細(xì)胞和feeder細(xì)胞的生長(zhǎng)能力。
相關(guān)形態(tài)學(xué)和分化分析：
檢測(cè)胎牛血清支持未分化ES細(xì)胞克隆的能力。通過(guò)堿性磷酸酶活性評(píng)估分化程度。未分化的ES細(xì)胞小顏色深紅粉紅，分化的細(xì)胞較大，豐滿(mǎn)，顏色較淺。
所有的分析在沒(méi)有ESGRO的情況下進(jìn)行的，培養(yǎng)基中出現(xiàn)ESGRO會(huì)掩蓋由胎牛血清所導(dǎo)致的問(wèn)題。[ESGRO或LIF經(jīng)常用來(lái)保持ES細(xì)胞處于未分化狀態(tài)。]
經(jīng)過(guò)培養(yǎng)發(fā)現(xiàn)，大約8批中有1批可以用來(lái)培養(yǎng)ES細(xì)胞。
26.在重新凍存sf9細(xì)胞前，它可以傳多少代？隨著傳代的次數(shù)的增加，它的感染能力會(huì)降低嗎？
通常情況當(dāng)細(xì)胞經(jīng)過(guò)30次傳代后，應(yīng)該返回凍存。無(wú)論什么時(shí)候記數(shù)時(shí)，都應(yīng)該檢查細(xì)胞活力。如果超過(guò)95％的細(xì)胞保持有活力和在大約30小時(shí)左右加倍，細(xì)胞仍然可以使用。如果活力和加倍時(shí)間下降，它們的感染力將不在是有效的。
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How do I choose the right medium?
There is no fixed culture condition for a certain type of cell culture. Cells cultured in MEM are likely to grow easily in DMEM or M199. In a word, MEM is the first choice for adhesion cell culture, RPMI-1640 for suspension cell culture, and AIMV medium [SFM] is the first choice for serum-free culture.
2. why heat inactivated serum?
Heating can inactivate the complement system. Activated complements participate in lysis events, stimulate smooth muscle contraction, release histamine from cells and platelets, and activate lymphocytes and macrophages. Heat-inactivated serum is recommended for immunological studies and culture of ES cells, insect cells and smooth muscle cells.
Is 3.L- glutamine important in cell culture? Is it unstable in solution?
L- glutamine is important in cell culture. L-glutamine can be used as a source of energy for cultured cells after removing amino acids, and it is involved in protein synthesis and nucleic acid metabolism. L-glutamine will be degraded after a period of time in solution, but the exact degradation rate has not been determined. L- degradation of glutamine leads to the formation of ammonia, and ammonia is toxic to some cells.
What is 4.GlutaMAX-I? How can cultured cells use GlutaMAX-I? How stable is the two peptide?
GlutaMAX-I dipeptide, a derivative of L-glutamine, protects its unstable alpha-amino group with L-alanine. A peptidase gradually cleavages two peptides and releases L- glutamine for utilization.
GlutaMAX-I dipeptide is very stable, even in 121 pounds sterilization for 20 minutes, GlutaMAX-I dipeptide solution has the smallest degradation, if under the same conditions, L-glutamine almost completely degraded.
5. why can we save phenol red in the medium?
Phenol red is used as a pH indicator in the medium: neutral red, acidic yellow, and basic purple. Studies have shown that phenol red can mimic steroid hormones (especially estrogen). To avoid steroid reaction, cells, especially mammalian cells, are cultured in a medium without phenol red. Because phenol red interferes with detection, some researchers do not use a culture medium containing phenol red for flow cytometry.
6. how to count live cells with trypan blue?
Cell suspension was diluted to 200-2000 cells per milliliter in serum-free medium and 0.4% Trypan Blue solution was added to 0.1 milliliter cells. After mixing for a few minutes, count the cells with a blood count plate. Living cells repel trypan blue, so the dying blue cells are dead cells.
7. how to eliminate pollution from tissue culture?
When it is important to cultivate pollution, researchers may try to eliminate or control pollution. First, identify the contaminants as bacteria, fungi, mycoplasma or yeast, isolate the contaminated cells from other cell lines, disinfect the culture dishes and super-clean tables with laboratory disinfectants, and check the HEPA filter.
High concentrations of antibiotics and antimycetin may be toxic to some cell lines, so dose-response tests are performed to determine the level of toxicity of antibiotics and antimycetin. This is especially important when using antibiotics such as amphotericin B and antifungal agents such as tylosin. The following are recommended steps for determining toxicity levels and eliminating contamination.
1) Digestion, counting and dilution of cells in antibiotic-free medium, dilution to the concentration of conventional cell passage.
2) disperse cell suspension into porous culture plate or several small culture bottles. Within a concentration gradient, the antibiotics are added to each pore. For example, amphotericin B recommends the following concentration, 0.25, 0.50, 1, 2, 4.0,8.0mg/ml.
3) observe cytotoxicity indicators every day, such as abscission, vacuolation, confluence reduction and roundness.
4) After determining the level of antibiotic toxicity, the cells were cultured for 2-3 generations in the medium containing 2-3 times the concentration of antibiotics.
5) cells were cultured in a medium without antibiotic.
6) repeat step 4.
7) culture the 4-6 generation in an antibiotic free medium to determine whether contamination has been eliminated.
8. what is the role of sodium pyruvate in culture medium?
Sodium pyruvate can be used as an alternative carbon source in cell culture. Although cells tend to use glucose as a carbon source, they can also metabolize sodium pyruvate without glucose.
9.Hank 's equilibrium salt solution [HBS] is used in air, and no CO2 incubator is required. What is the reason? What are the essential functional differences between Hank's equilibrium salt solution [HBS] and Earle's equilibrium salt solution [EBS]?
The main difference between HBS and EBS is the level of sodium bicarbonate, which is higher in Eagles (2.2g/L) than in Hanks (0.35g/L). Sodium bicarbonate requires a high level of CO2 balance to maintain the pH value of the solution. Eagles solution will become alkaline in CO2 at air level, and Hanks solution will become acidic in CO2 incubator. If you want to preserve tissue in CO2 incubator, you need Eagles solution. If you just clean the tissue that will be stored in the cell culture medium, you can use Hanks solution.
What is the difference between 10.Qualified and Certified fetal bovine serum?
Certified fetal bovine serum contains all the standard tests performed by Qualified fetal bovine serum, and in addition to these standard tests, Certified fetal bovine serum has the following additional tests:
End-PointDeterminationofEndotoxinContent
Bacteriophage test
Biochemical detection
Detection of hormones
Hemoglobin detection
Sf9 cell growth promotion and methodological detection
11. can two valent ions inhibit trypsin activity? What is the purpose of adding EDTA to trypsin?
Two valence ions do inhibit trypsin activity. EDTA is used to chelate free magnesium and calcium ions in order to maintain the inhibitory activity of trypsin. It is suggested that the cells should be washed with EDTA before trypsin treatment to remove all the divalent ions from the medium.
12. will the preparation of lipid-DNA affect the transfection efficiency?
Yes. For LIPOFECTAMlNE Reagent, the diluent reagent diluted DNA in 100 ml OPTI-MEM and 100 ml OPTI-MEM. The mixture was incubated for two minutes at room temperature for 15 minutes. For LIPOFECTIN Reagent, allowing diluted reagents and media to incubate for at least 30 minutes before adding DNA solution can triple the efficiency. Make sure that the complex is formed without serum. After incubation for 15 minutes, the medium containing [800 was added to the compound l]. [note that the volume of 35mm dish is used]. For LIPOFECTAMlNEPlus Reagent DNA, it should be mixed with Plus Reagent before mixing with liposomes. The LIPOFECTAMlNE2000 procedure allows the mixing of DNA and liposomes in a very small volume, which can then be added directly without changing the medium.
13. When I used SF900II, the cells grew well, but why was my protein product less effective than Graces solution and 10% fetal bovine serum?
If the target protein is a late-stage protein, it will be expressed with proteases that act on the target protein. In a serum-supplemented medium, these proteases act on the proteins in the serum to keep the target protein product intact. In serum-free formulations, the only substrate for protease action is your target protein. To avoid this problem, some protease inhibitors or a small amount of serum [less than 1%] were added to give the protease a substrate.
14. how to detect the level of endotoxin (heat source)?
The LAL [Limulus Amebocyte Lysate] test is the most sensitive and specific method available for the detection of bacterial endotoxins. Levin and Bang found that bacteria could cause coagulation in the Limulus [Limuluspolyphemus] vessels. Endotoxin starts an intracellular enzymatic system [serine protease cascade reaction system], which modifies lectins to produce a transparent gum, coagulation protein in LAL, and thus forms insoluble matrices. LAL reagent buffered horseshoe crab lysates.
The fetal bovine serum endotoxin test was carried out on Grand Island using the Gel-clot method in the manual. Before the endotoxin test was carried out, the samples were diluted with water without heat source at 1:10. The diluted sample was boiled for 5 minutes to remove the inhibitor. [Usually, the presence of endotoxin-binding components in the blood inhibits the gel process and prevents endotoxin from reacting with LAL. This inhibition can be eliminated by pre heat treatment. ]
15. can I use a solid form of MurashigeSkog medium?
If solid medium is used, agar should be added. The agar should be sterilized before adding. Direct sterilization of MS medium should be avoided. Agar solution should be autoclaved and then melted agar solution should be added to MS medium.
Will the pH change at 16.20 or higher temperatures at higher or lower temperatures?
For ordinary buffers, pH changes with temperature.
The following table shows the change of pH value when the temperature changes 10 degrees.
For example, the PH7.4Tris buffer is prepared at 20 degrees centigrade, and the pH value is 7.4-[2x0.310] = 6.78 at 40 C.
17. what is the pH value of Tris-HCl solution prepared at room temperature (25 C) at 37?
The pH of the buffer varies with temperature. The following table shows the different pH values of 50mMTris-HC solution at 4, 25 and 37 C.
18. what is the optimum pH and osmotic pressure of insect cell culture?
The PH value of the growth medium can affect the cell proliferation and the production of virus or recombinant protein. For most of the insect cell lines of Lepidoptera, the application effect was good in the range of PH 6.0-6.4. The optimum osmotic pressure of medium was 345-380mOsm/kg. when cultured lepidopteran cell lines. To ensure reliable and sustained cell culture, reduce technical problems, maintain PH values and osmotic pressure in the range listed above.
Are there any other names for 19.HighFive cells?
HighFive cells are also known as Trichoplasiani5B1-4 and BTI-TN-5B1-4.
20. what is the concentration of detergent in HighFive serum-free medium?
The concentration of detergent in HighFive serum-free medium was 0.025g/LTween-80 and 1.0g/LPluronic Poly-all.
How much density does 21.HighFive cells cryopreservation?
3.0x10E6cells/ml
22. a white precipitate was found in my Drosophila medium and dissolved after heating. What is it? Is it harmful to my cells?
It may be glutamine precipitation, but more likely it is L- tyrosine precipitation. The concentration of glutamine in culture medium is 6 times higher than that in typical 2mM. The concentration of tyrosine is 25 times higher than that in RPMI1640, and it is more difficult to dissolve than glutamine. Precipitation may also be a compound of more than one component. It may be caused by low temperature. As long as the precipitates can be dissolved under culture conditions, there will be no adverse effects on the experiment.
23. how to transfer Sf9 cells from T25 bottles? Can it be digested with trypsin?
We strongly recommend the use of exfoliated cells because this technique is the least destructive and most viable. By using the pasteurized pipette, the cell medium can be moved. As an option, you can gently tap the culture bottle. Trypsin is used to digest cells only when absolutely necessary.
Trypsin digestion of Sf9 cells from a T25 bottle:
1) remove the culture medium.
2) 2ml1xPBS[is enough to cover the cell surface] scrub cells and remove PBS.
3) trypsin EDTA[was added to the 2ml1x to cover the cell surface.
4) incubated for 37 to 5 minutes to 10 minutes. They are moving up 5 minutes after testing.
5) 2 ml cell culture medium was added into the cells, and then transferred into the conical tube. The flask wall was washed with 2 ml culture medium and transferred into the same conical tube. [FBS in culture medium terminated the activity of pancreatin. ]
6) centrifugally [1100rpm] precipitated cells. Remove the medium.
7) resuspension cells with new medium. Passages.
24. what is the usage of heparin in Sf9, Sf21, and highFive cell suspension cultures?
To prevent cell aggregation in suspension cultures, heparin concentrations of 10 units/ml were used.
25. how to assess the qualified fetal bovine serum of ES cells?
Use D3ES cells. This is a cell line sensitive to growth-promoting, growth-inhibiting and differentiation factors in fetal bovine serum.
Related growth efficiency analysis:
When ES cells were introduced into a growth medium containing 10% fetal bovine serum at a very low density, the ability to initiate and support ES cell cloning was tested.
Cytotoxicity analysis:
The growth ability of ES cells and feeder cells was tested when they were introduced into the growth medium containing 30% fetal bovine serum at very low density.
Related morphology and differentiation analysis:
The ability of fetal bovine serum to support the cloning of undifferentiated ES cells was detected. The degree of differentiation was assessed by alkaline phosphatase activity. The undifferentiated ES cells were dark red and pink in color, and the differentiated ES cells were large, plump and light in color.
All analyses were performed without ESGRO, and the presence of ESGRO in the medium masked problems caused by fetal bovine serum. [ESGRO or LIF are often used to maintain ES cells in an undifferentiated state. ]
After training, 1 batches of about 8 batches can be used to culture ES cells.
26. how many generations can it pass before re cryopreservation of Sf9 cells? As the number of passages increases, will its infection ability decrease?
Usually, after 30 passages, the cells should be returned to cryopreservation. Whenever you count your numbers, you should check cell viability. If more than 95% of the cells remain viable and doubled in about 30 hours, the cells can still be used. If vitality and double time fall, their appeal will not be effective.