有限稀釋法是一種常用的克隆方法，是指將需要再克隆的細胞株自培養(yǎng)孔內(nèi)吸出并作細胞計數(shù)，計出1mL的細胞數(shù)。 有限稀釋法原理 有限稀釋的操作原理為將一定溶度的溶液進行倍比稀釋。例如準備8個試管，第一個管中加入150ml培養(yǎng)液，余下七個試管加入100ml buffer，從第一試管去50ml加入第二試管，充分混勻后取50ml加入第三試管，依次類推。則每一試管的濃度較上一試管相比稀釋了3倍，由此形成了一個有8個濃度梯度的倍比稀釋溶液。 有限稀釋法操作流程 有限稀釋法的具體實驗操作流程有多種，本文就對搜尋到的三種操作流程進行介紹： 一、 1.1材料 （1）微量培養(yǎng)盤，盤內(nèi)各孔于克隆化前一天培養(yǎng)小鼠腹腔細胞（即飼養(yǎng)細胞）每孔2萬～4萬。 （2）HT培養(yǎng)基 1.2操作方法 （1）取出抗體陽性孔細胞，用HT培養(yǎng)液制成細胞懸液。并取樣進行臺盼蘭染色，計數(shù)。 （2）用HT培養(yǎng)液將細胞稀釋成200個/ml、40個/ml、20個/ml和的懸液。 （3）用吸管將細胞懸液分別種入微量培養(yǎng)盤，每孔0.05ml，細胞含量分別為10個/孔、2個/孔、1個/孔和0.5個/孔。 （4）5％CO2飽和濕度，37℃培養(yǎng)。 （5）每天用倒置顯微鏡觀察克隆生長情況，選擇只有一個集落生長的孔，棄掉兩個以上和沒有細胞生長的孔。 （6）克隆大量繁殖后，布滿孔底的1/3～1/2時，測培養(yǎng)液抗體。 （7）抗體陽性孔細胞，移到有飼養(yǎng)層的組織培養(yǎng)瓶中，并傳2～4代就可以脫離飼養(yǎng)細胞，建成克隆株。 二、 1、取對數(shù)生長期的細胞，胰酶消化后制成單個細胞懸液。 2、取少量細胞懸液，用臺盼藍染色并計數(shù)活細胞。 3、將細胞懸液移至刻度離心管中連續(xù)倍數(shù)稀釋。稀釋到10個細胞/ml。 4、將稀釋好的細胞懸液接種到96孔板中，每孔加液0.1ml。然后放入培養(yǎng)箱內(nèi)培養(yǎng)。 5、次日，在倒置顯微鏡下觀察培養(yǎng)板各孔細胞數(shù)，挑選只含一個細胞的孔（由于細胞懸液并不完全均一，所以有2-3個細胞/孔，也有沒有細胞的孔），做好標記并補加0.1ml培養(yǎng)液繼續(xù)培養(yǎng) 說明：第一種方法與第二種方法的區(qū)別在于第一種方法會稀釋三種濃度梯度放入96孔板中培養(yǎng)，而第二種是先稀釋至10個/ml，再取0.1ml加入96孔板，從而保證每孔有一個細胞。 三、 該法是作者在一篇外文文獻上找到的方法，采用了縱向與橫向兩個方向進行稀釋形成了兩個稀釋梯度（見下圖）

 

 

其培養(yǎng)后的結(jié)果為（見下圖）

 

 

具體的操作流程為： Supplies Nonsterile 1. Pipetting aids - Corning Cat. # 4910 (1) 2. Disposal tray or bucket for used pipettes (1) 3. Marking pen (1) 4. 200μL pipettor - Corning Cat. # 4963 (1) 5. 8-channel multichannel 200μL pipettor- Corning Cat. # 4888 (1) Sterile 1. Cell culture medium – (Appropriate culture medium for the cells that will be cloned) (30mL) 2. Cell suspension at 5x104 to 1x105 cells/mL (200μL) 3. 96 well cell culture plate - Corning Cat. # 3585 (1) 4. Sterile pipettor tips 5. Reagent dispensing reservoir/tray - Corning Cat. # 4870 or 4871 (1) 6. 1, 5, and 10mL pipettes - Corning Cat. # 4485, 4487 and 4488 Procedure 1. Fill the reagent dispensing tray with 12mL of the appropriate culture medium, then using the 8 channel pipettor add l00μL medium to all the wells in the 96 well plate except well Al (see diagram below) which is left empty 2. Add 200μL of the cell suspension to well A1. (See Figure 1.) Then using the single channel pipettor quickly transfer 100μL from the first well to well B1 and mix by gently pipetting. Avoid bubbles. Using the same tip, repeat these 1:2 dilutions down the entire column, discarding 100μL from H1 so that it ends up with the same volume as the wells above it. 3. With the 8-channel pipettor add an additional l00μL of medium to each well in column 1 (giving a final volume of cells and medium of 200μL/well). Then using the same pipettor quickly transfer l00μL from the wells in the first column (Al through H1) to those in the second column (A2 through H2) and mix by gently pipetting. Avoid bubbles. 4. Using the same tips, repeat these 1:2 dilutions across the entire plate, discarding l00μL from each of the wells in the last column (A12 through H12) so that all the wells end up with 100μL of cell suspension. 5. Bring the final volume of all the wells to 200μL by adding 100μL medium to each well. Then label the plate with the date and cell type. Incubate plate undisturbed at 37°C in a humidified CO2 incubator. 6. Clones should be detectable by microscopy after 4 to 5 days and be ready to score after 7 to 10 days, depending on the growth rate of the cells. Check each well and mark all wells that contain just a single colony. These colonies can then be subcultured from the wells into larger vessels. Usually each clone is transferred into a single well in a 12 well or 24 well plate.