哺乳動物生物發(fā)光，一般是將Firefly luciferase基因（由554個氨基酸構成，約50KD）即熒光素酶基因整合到預期觀察的細胞染色體DNA上以表達熒光素酶，培養(yǎng)出能穩(wěn)定表達熒光素酶的細胞株，當細胞分裂、轉移、分化時, 熒光素酶也會得到持續(xù)穩(wěn)定的表達。基因、細胞和活體動物都可被熒光素酶基因標記。將標記好的細胞接種到實驗動物體內后，當外源（腹腔或靜脈注射）給予其底物熒光素(luciferin)，即可在幾分鐘內產生發(fā)光現象。這種酶在ATP，氧存在的條件下，催化熒光素的氧化反應才可以發(fā)光，因此只有在活細胞內才會產生發(fā)光現象，并且發(fā)光光強度與標記細胞的數目線性相關。
熒光素由于諸多優(yōu)點得到廣大科研人員的青睞，主要特點如下：
(1) 熒光素不會影響動物的正常生理功能。
(2)熒光素是280道爾頓的小分子，水溶性和脂溶性都非常好，很容易穿透細胞膜和血腦屏障。
(3) 熒光素在體內擴散速度快，可通過腹腔注射或尾部靜脈注射進入動物體內。腹腔注射擴散較慢，持續(xù)發(fā)光長。熒光素腹腔注射老鼠后約1min后表達熒光素酶的細胞開始發(fā)光，10min后強度達到穩(wěn)定的最高點，在最高點持續(xù)約20~30 min后開始衰減，約3h后熒光素排除，發(fā)光全部消失，最佳檢測時間是在注射后15~35min之間；若進行熒光素靜脈注射，擴散快，但發(fā)光持續(xù)時間很短。科研人員根據大量的實驗總結出熒光素的合適的用量是150mg/kg，即體重20克的小鼠需要3毫克的熒光素。
(4) 觀察時間的間隔沒有最短限制，只要觀察的條件控制一致就可以。雖然底物在動物體內有一定的代謝過程，但是上一次底物的殘留曲線可以知道，可以控制對下一次觀察結果的影響。
光學原理：光在哺乳動物組織內傳播時會被散射和吸收，光子遇到細胞膜和細胞質時會發(fā)生折射現象，而且不同類型的細胞和組織吸收光子的特性并不一樣。血紅蛋白（hemoglobin）是造成體內可見光被吸收的主要因素，其吸收可見光中藍綠光波段的大部分。但是在可見光大于600納米的紅光波段，血紅蛋白的吸收作用卻很小。因此，在偏紅光區(qū)域, 大量的光可以穿過組織和皮膚而被檢測到。利用活體動物生物發(fā)光成像技術最少可以檢測到皮下的幾百個細胞。當然，由于發(fā)光源在老鼠體內深度的不同可看到的最少細胞數是不同的。一般認為，每一厘米深度，發(fā)光強度衰減10倍，血液豐富的組織或器官（比如心臟、肝臟、肺臟）衰減多，與骨骼相鄰的組織或器官衰減少。在相同的深度情況下，檢測到的發(fā)光強度和細胞的數量具有非常好的線性關系，可由儀器量化檢測到的光強度，反映出細胞的數量。
熒光素酶的發(fā)光是生物發(fā)光，不需要激發(fā)光，但需要底物熒光素(D-Luciferin,鉀鹽)。熒光素酶有554個氨基酸，約50KD。熒光素酶的底物熒光素，約280道爾頓。熒光素的水溶性和脂溶性都非常好，很容易穿透細胞膜和血腦屏障。熒光素是腹腔注射或尾部靜脈注射進入小鼠體內的，約一分鐘就可以擴散到小鼠全身。大部分發(fā)表的文章中，熒光素的濃度是150mg/kg 。20克的小鼠需要3毫克的熒光素。常用方法是腹腔注射，這種方法擴散較慢、開始發(fā)光慢、持續(xù)發(fā)光長。若進行熒光素靜脈注射，這種方法擴散快、開始發(fā)光快，但發(fā)光持續(xù)時間很短。熒光素(luciferin)在氧氣、ATP存在的條件下和熒光素酶發(fā)生反應，生成氧化熒光素(oxyluciferin)，并產生發(fā)光現象。熒光素不會影響動物的正常生理功能。我們提供1g，400mg以及200mg 的包裝，價格優(yōu)惠。
常用的有兩種熒光素酶，Luciferase 和 Renilla熒光素酶，二者的底物不一樣，前者的底物是D-luciferin，后者的底物是coelentarizine （我們也有，如果需要請聯系）。二者的發(fā)光顏色不一樣，前者所發(fā)的光波長在540-600nm，后者所發(fā)的光波長在460-540nm左右。前者所發(fā)的光更容易透過組織，后者在體內的代謝比前者快。大部分的發(fā)表文章通常使用前者用作報告基因， 也有一些文章使用兩者進行雙標記。
熒光素腹腔注射老鼠后約一分鐘后表達熒光素酶的細胞開始發(fā)光，十分鐘后強度達到穩(wěn)定的最高點。在最高點持續(xù)約20-30 分鐘后開始衰減，約三小時后熒光素排除，發(fā)光全部消失。最好的檢測時間是在注射后15到35分鐘之間。
熒光素酶基因是插到細胞染色體內的，當細胞分裂、轉移、分化時, 熒光酶也會得到持續(xù)穩(wěn)定的表達。熒光素酶的半衰期約三個小時, 所以只有活細胞才能夠持續(xù)表達熒光素酶。
實驗方法如下：
Protocol 1: In Vitro Imaging of MDA-MB-231-luc and HCT116-Luc cells
Materials needed:
MDA-MB-231-luc cells (1 T175 flask)
HCT116-Luc (1 T75 flask)
Trypsin/EDTA
D-Luciferin Firefly, potassium salt（Sciencelight, Catalog# luc001）, 1.0 g /vial, 
Sterile water
Complete media for each cell line
96 well black plates (Costar)
Procedure:
A. Luciferin Preparation
1.  Prepare a 200X luciferin stock solution (30 mg/ml) in sterile water.  Mix gently by inversion until luciferin is completely dissolved. Use immediately, or aliquot and freeze at -20C for future use.
* Note: Reconstitute the quantity of D-Luciferin necessary for an individual experiment.
2.  Prepare 2X luciferin (300ug/ml) in complete media.  Quick thaw 200X stock solution and dilute 1:100 in complete media (2X solution)
                * Note: This working solution should be prepared fresh for each experiment.
B. Cell Preparation and Serial Dilution
1.  Trypsinize a flask and resuspend cells in a total of 10ml media.
2.  Count cells and adjust cell concentration to 100,000 cells/ml = 10,000 cells/100ul.
3.  Perform a serial dilution (1:2) in complete media as follows:
        a.  Pipette 200 ul cells into well #1 (in duplicate).
        b.  Add 100 ul media to wells #2-10 (in duplicate) using a multi-channel pipette.
c.  Perform 1:2 serial dilution by pipetting 100ul from well #1 to well #2 using a multi-channel pipette, mixing well, then taking 100 ul from well 2 to well 3.  Continue dilution to well #10 and discard 100 ul from well #10 after mixing.
C.  Imaging Procedure
1.  Add 100 ul 2X luciferin to cells in well #1-10.
2.  Place plate of cells in the camera system on FOV15cm to view entire plate.  Use the Plate Positioner or Alignment Grid to help position the plate.  
3.  Approximately 2-3 minutes after the addition of luciferin, image plate at 1 minute, 10 bin.
        * Note: If using >10,000 cells in well #1, imaging time should de decreased.
D. Data Analysis
1.  Apply grid ROI to image; measure photons/well.
2.  Export measurements from LI lab book to an excel file.
3.  Are dilutions consistent?  What is the minimum number of cells detected?
Protocol 2: In Vivo imaging of MDA-MB-231-luc and HCT116-Luc Subcutaneous Model
Materials:
MDA-MB-231-Luc and HCT116-Luc cells
Mice: 2 subcutaneous HCT116-Luc tumor-bearing female BALB/c nude mice;
4 naïve female BALB/c nude mice;
Artagain black paper (Strathmore, Catalog# 445-109)
D-Luciferin Firefly, potassium salt（Sciencelight, Catalog# luc001）, for in vivo imaging
Syringe (1ml) and needles (25 x 5/8" gauge)
Anesthesia (Pentobarbital Sodium)
Procedure:
A.        Luciferin Preparation
1.        Prepare a stock solution of luciferin at 15mg/ml in DPBS. Filter sterilize through a 0.2 um filter.
2.        Prepare enough to inject 10 ul/g of body weight.  Each mouse should receive 150 mg luciferin/kg body weight.  (e.g. For a 20 g mouse, inject 200 ul to deliver 3 mg of luciferin.)
B.         Preparation of tumor cells:
1.        Trypsinize the cells from flasks, wash once in serum-free medium.
2.        Resuspend in room temperate serum-free medium to a concentration of 1x106 cells in 100ul.  Hold at room temperature until ready for use.
C.        Injections:
1.        Inject mice with firefly luciferin (150 mg/kg) by intraperitoneal injection using a 25 x 5/8” gauge needle.  
2.        After 7-8 minutes, anesthetize mice by intraperitoneal injection of pentobarbital sodium using a 25 x 5/8” gauge needle.  
3.        (1) Inject tumor cells (1x106 cells in 100ul) subcutaneously into the dorsal flank of the mouse. 
     (2) For the two HCT116-Luc tumor-bearing mice, no cells were inoculation to the mice.
D.        Imaging
Place mice on black paper in the imaging box and image dorsally to detect tumor cells.
Protocol 3:  In Vivo imaging of HCT116-Luc Intravenous Model
Materials:
HCT116-Luc cells
Mice: 1 naïve female BALB/c nude mice
Artagain black paper (Strathmore, Catalog# 445-109)
D-Luciferin Firefly, potassium salt（Sciencelight, Catalog# luc001）, for in vivo imaging
Syringe (1ml) and needles (25 x 5/8" gauge and 26 x 1/2" gauge)
Heating lamp and mouse restrainer (for cell injection)
Anesthesia (Pentobarbital Sodium)
Procedure:
A.        Luciferin Preparation
1.        Prepare a stock solution of luciferin at 15mg/ml in DPBS. Filter sterilize through a 0.2 um filter.
2.        Prepare enough to inject 10 ul/g of body weight.  Each mouse should receive 150 mg luciferin/kg body weight. (e.g. For a 20 g mouse, inject 200 ul to deliver 1.5 mg of luciferin.)
B.         Preparation of tumor cells:
3.        Trypsinize the cells from flasks; wash once in serum-free medium.
4.        Resuspend in room temperate serum-free medium to a concentration of 1x106 cells in 100ul.  Hold at room temperature until ready for use.
C.        Injections:
1.        Inject mice with firefly luciferin (150 mg/kg) by intraperitoneal injection using a 25 x 5/8” gauge needle.  
2.        Expose mice to heating lamp for tail vein dilation (4-5 minutes).
3.        Place mice into a restrainer and inject tumor cells (1x106 cells in 100ul) SLOWLY i.v. through the tail vein using a 26 x 1/2” gauge needle. 
4.        Anesthetize mice by intraperitoneal injection of pentobarbital sodium using a 25 x 5/8” gauge needle.  
D.         Imaging:
        Mice are placed onto black paper in the imaging box and imaged ventrally.