蛋白质磷酸化与信号传导

蛋白质磷酸化与信号传导

蛋白質磷酸化與信號傳導

對所有真核生物而言，蛋白質的磷酸化是細胞內信號傳遞的關鍵步驟，舉凡在細胞代謝、生長、增殖、癌變調控方面，蛋白質的磷酸化都扮演著重要角色。目前在真核細胞內已發現有400多種蛋白激酶和1000多種磷酸蛋白。通過蛋白質磷酸化，機體得以改變細胞內蛋白質活性，或藉以改變酵素活性、或藉以傳遞信號、或調節細胞內各個生理過程。隨著愈來愈多的信號傳導路徑被證實與蛋白質磷酸化相關，此一主題己是當今細胞生物學研究的熱點。

蛋白質的可逆磷酸化

蛋白質的磷酸化和去磷酸化是一個可以相互轉化的過程，分別由機體藉由調控細胞內各類蛋白激酶（protein kinases）和蛋白磷酸酶（phosphatases）的活性來啟動或抑制。通過磷酸化和去磷酸化兩種構象的互變，導致蛋白質活性改變而表達其特殊的生理功能。現已證實，細胞的許多功能蛋白，如激酶、酶蛋白、受體、視蛋白、運輸蛋白、收縮蛋白、調節蛋白、核蛋白、離子通道、離子幫浦等，均能發生蛋白質的磷酸化與去磷酸化。蛋白質磷酸化的方式有兩種：一種是蛋白質自身磷酸化，如胰島素受體、C激酶、G激酶、Ca2+·CaM激酶、EGF受體、PDGF受體、IGF-1受體、FGF受體、CSF-1受體、Na+-K+-ATP酶的α亞基、Ca2+-ATP酶的磷酸化等。另一種是由特殊的蛋白激酶催化的蛋白質磷酸化。蛋白質磷酸化的部位可發生在蛋白質的絲氨酸、蘇氨酸、酪氨酸、賴氨酸、組氨酸及天門冬氨酸的殘基上，使磷酸化蛋白質發揮著多種多樣的生物學功能。

蛋白质磷酸化与信号传导

細胞內還存在專一性蛋白磷酸酶。這種酶特異地催化酪氨酸和絲氨酸磷蛋白去磷酸化，通過去磷酸化改變多種功能蛋白（激酶、酶、受體、運輸蛋白、收縮蛋白、離子幫浦、離子通道、視蛋白、調節蛋白、核蛋白等）的活性，從而調節細胞的代謝、生長、分化、增殖及轉化，在細胞內生物資訊的傳遞中起極其重要的作用。

新技術：單一細胞之信號傳導

流式分析的主要特色，在於可以同時分析單一細胞的多種特徵，當同時使用多種分子探針，與表面標記，可同時識別出特定的細胞亞群，如Ｔ淋巴細胞(CD3+)、B淋巴細胞(CD19+)、NK細胞，並告知該群細胞族群的胞漿內磷蛋白，是否因外在淚活而被磷酸化？是否能因加入信號傳遞抑制物而去磷酸化？

傳統方法如西方墨漬法，由於屬於大多數分析(Bulk Assay)，一般都預設群體中大多數細胞的作用趨勢相同，作用強度一致，無法顯示異質化群體間的個別差異，如% Responder，實驗報告也只能顯示整體細胞的中間值，無法提供單一細胞的測量值。

研習會

本研習會以周邊血淋巴細胞的啟動為例，來介紹「蛋白質磷酸化」流式分析方法。淋巴細胞的啟動包括一系列連續發生的事件：識別信號、啟動信號的跨膜傳遞、胞內信號傳遞、DNA結合蛋白的活化和轉位、基因的轉錄啟動、新分子的表達、細胞因子的分泌、進入細胞周期等等。

應用phorbol myristate ester（PMA），或合併應用鈣離子導入劑A23187來刺激淋巴細胞，可直接啟動胞漿中信號傳遞途徑中的重要成份Protein Kinase C（PKC），迅速而有效地激活T細胞。PKC可接著活化 Raf - MEK – ERK 信號途徑（如例圖），提供本示範實驗的陽性對照。

蛋白质磷酸化与信号传导

抗癌藥物開發之應用

不少癌細胞都有表現異常高的蛋白質磷酸化現象，這種變化很可能是引起細胞周期調控失常的主因，特別是在所謂Mitogen Activated Protein Kinase (MAP Kinase)信號途徑發生異常信號傳遞的頻率很高。發生的原因可能因為生長因子受體的變異，或Ras癌基因的活化。

多年來，癌細胞內各類異常活性的蛋白激酶和蛋白磷酸酶，一直是各藥廠研發人員有興趣的分子標靶，希望可以針對這些磷酸化蛋白質來設計出可控制癌細胞生長、分裂的藥物，來阻止癌細胞的增殖。國外藥廠目前運用這類原理的抗癌藥物有Met癌基因、CDK細胞周期激酶、CDC25蛋白磷酸酶等之分子抑制劑，並已有數個先驅藥物正進行腫瘤治療的臨床試驗。

信號傳導抑制藥理學研究

如果知道先驅藥物的分子標靶，是針對某特色的信號傳遞路徑，如UO126抑制MEK活性，BAY37-9751抑制Raf kinase活性，研發人員便可以用流式分析分段進行藥動學研究，在體外用細胞核型來研究劑量與時間之關係圖，或在臨床試驗抽血檢查病人之藥效。

蛋白质磷酸化与信号传导

Introducing BD PhosFlow Reagents

蛋白質磷酸化FCM分析試劑

BD Biosciences Pharmingen 提供最完整的蛋白質磷酸化分析試劑，配合既有的一系列表面ＣＤ標記，BD Transduction LaboratoriesTM 讓研究人員可隨心所欲地設計實驗，來瞭解細胞內複雜交錯的信號傳導途徑。特別介紹BD Transduction LaboratoriesTM 系列產品，多色的直接螢光標記，可搭配進行3-、4-、5-、甚至6色螢光分析。

探索細胞儀的強效分析力

可在單一實驗中，同時觀察不同的磷酸化事件，如Caveolint(pY14), ERK 1/2 (pT202/pY204), P38 MAPK (pT180/pY182), Stat1 (pY701), Stat3 (pY705), Stat5 (pY694), Stat6 (pY641)等。可在異質化細胞該群中，圈選出特定該群來分析其胞漿內磷蛋白之磷酸化程度。

蛋白质磷酸化与信号传导

LABORATORY PROTOCOLS 一、試劑、材料： y y y y y y y y

Ficol-Hypaque or CPT tubes

RPMI 1640 medium containing 10% fetal calf serum （pre-warm to 37℃）

40uM PMA (Sigma #P8139; phorbol myristate acetate 40uM working dilution in ethanol) 10% formaldehyde (Ultrapure EM Grade; Polysciences) （pre-warm to 37℃） FACS staining buffer (1x PBS, 1% FBS, 0.09% NaN3) （pre-warm to room temp） BD PhosFlow Antibodies to ERK1/2（BD Pharmingen cat # 612566） BD Anti-Human CD3 Antibody（BD Pharmingen cat # 556611） Phosphate Buffered Solution（pre-warm to 37℃）

y 100% Methanol（Ice cold）

二、 檢體收集、處理：

Peripheral blood samples using EDTA as the anticoagulant were obtained from normal donors. Mononuclear cell suspensions were obtained by density gradient centrifugation on

Ficol-Hypaque, and the concentration adjusted to 1x106/ml in RPMI 1640 medium containing 10% fetal calf serum.

三、 PMA激活淋巴細胞內ERK及MEK

Resuspend cells at 1x106/ml in tissue culture medium such as RPMI 1640 plus 10% fetal bovine serum (FBS), 37° water bath. Activate for 10 min using 40nM PMA (final concentration). Also prepare non-activated control cells using mock treatment.

四、應用BD PhosFlow 流式分析試劑進行直接螢光染色

1. Collect experimental (activated) and control cells in separate polystyrene tubes, pellet by

centrifugation (250 x g) for 5 minutes, and discard supernatant. Resuspend the pellet with 4ml PBS.

蛋白质磷酸化与信号传导

2. While maintaining sample at 37°, fix by adding 1ml 10% formaldehyde to give a final

concentration of 2% formaldehyde. Incubate cells at 37℃ for 10 minutes. 3. Wash cells once with 5 ml FACS staining buffer (1x PBS, 1% FBS, 0.09% NaN3). 4. Resuspend cells in staining buffer at 10 x 106 cells/ml.

5. Aliquot 100 μl of cells (~1×106 cells/ml) to each tube. Perform surface staining by adding

10 μl FITC-conjugated Anti-Human CD3 monoclonal antibodies. Incubate in the dark for 30 minutes at room temperature. NOTE: Some antibodies which recognize native cell surface markers may not bind to fixed/denatured antigen or signal intensity can be decreased. We do not routinely test cell surface antibodies on fixed/permeablized cells. 6. Wash cells once with 3ml staining buffer (2ml/wash for staining in tubes), pellet by

centrifugation (250 x g), and discard supernatant.

7. Permeabilize cells by thoroughly resuspending in 0.5 ml cold 90% methanol. Incubate for

30 minutes on ice.

8. Wash once with 3 ml staining buffer, pellet by centrifugation (250 x g) for 5 minutes and

discard supernatant.

9. Perform intracellular staining by resuspending fixed/permeabilized cells in 100 μl of

staining buffer. Add 10 μl of PE-conjugated, phospho-specific Anti-ERK1/2 antibodies. Mix well and incubate at room temperature for 1 hour in the dark.

10. Wash once with 3ml staining buffer. Resuspend in 0.5 ml staining buffer prior to flow

cytometric analysis.

11. Run on flow cytometer. Flow cytometry was done using an FACScan analyzer (Becton

Dickinson, San Jose, CA). An air-cooled argon laser operating at 15mW was used for excitation. Fluorescence signals were collected using bandpass filters centered at 530nm and 585nm, with fluorescence compensation set to suppress FITC spillover into the PE channel.

五、間接螢光染色技術

1. Collect experimental (activated) and control cells in separate polystyrene tubes, pellet by

centrifugation (250 x g) for 5 minutes, and discard supernatant. Resuspend the pellet with 4ml PBS.

2. While maintaining sample at 37°, fix by adding 1ml 10% formaldehyde to give a final

concentration of 2% formaldehyde. Incubate cells at 37℃ for 10 minutes.

蛋白质磷酸化与信号传导

3. Wash cells once with 5 ml FACS staining buffer (1x PBS, 1% FBS, 0.09% NaN3). 4. Resuspend cells in staining buffer at 10 x 106 cells/ml.

5. Aliquot 100 μl of cells to each tube. Perform surface staining by adding fluorochrome-

conjugated monoclonal antibodies specific for cell surface antigens . Incubate in the dark for 30 minutes at room temperature.

6. Wash cells once with 3ml staining buffer, pellet by centrifugation (250 x g), and discard

supernatant.

7. Permeabilize cells by thoroughly resuspending in 0.5 ml cold 90% methanol. Incubate for

30 minutes on ice.

8. Wash once with 3 ml staining buffer, pellet by centrifugation (250 x g) for 5 minutes and

discard supernatant.

9. Perform intracellular staining by resuspending fixed/permeabilized cells in 100 μl of

staining buffer containing 2 μl phospho-specific Anti-Erk 1/2 antibodies（BD Pharmingen cat# 612358）. Incubate at room temperature for 1 hour in the dark.

10. Wash once with 5 ml 1x staining buffer, pellet by centrifugation (250 x g) for 5 minutes and

discard supernatant.

11. Cells were then incubated with 2 μl PE-conjugated Rat anti-mouse-IgG1 antibody（BD

Pharmingen cat# 550083）for 30 minutes at room temperature in the dark. 12. Wash once with 5 ml staining buffer. Resuspend in 0.5 ml staining buffer prior to flow

cytometric analysis.

13. Run on flow cytometer. Flow cytometry was done using a FACScan analyzer (Becton

Dickinson, San Jose, CA). An air-cooled argon laser operating at 15mW was used for excitation. Fluorescence signals were collected using bandpass filters centered at 530nm and 585nm, with fluorescence compensation set to suppress FITC spillover into the PE channel.

六、預期結果、注意事項：

Effects of PMA is similar in CD3 positive and negative cells. Expect to see 20-30 fold increase in pERK1/2 and 10-20 fold increase in pMEK with PMA activation. Use isotype controls or omitting primary antibodies if needed. The specific MEK inhibitor U0126 (Cell Signal Technology #9903), added at a final concentration of 10mM for 15 min at 37° prior to PMA treatment, blocks the activation of pERK1/2 but not pMEK.

蛋白质磷酸化与信号传导

七、Western Blots

Protein extracts were prepared from isolated lymphocytes using lysis buffer (1% Triton X-100, 0.1% SDS, 50mM Tris (pH 8.0) 150 mM NaCl, 1mM phenylmethylsulfonyl fluoride, 0.1mM NaVO4, 0.1mM benzamidine, 5mg/ml leupeptin, and 5mg/ml aprotinin). Samples containing 25mg of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using the Mini Trans-Blot Electrophoresis Transfer Cell (Bio-Rad, Mississauga, ON). ERK1/2 activity was detected using the phospho-specific antibody obtained from BD Pharmingen. Secondary antibody containing the horseradish peroxidase detection system for chemiluminescence was used as recommended by the manufacturer.

References： 1.

Burkett PJ., Nelson BJ., et al. 2003 Intracellular analysis of activated cell signaling molecules through the use of phospho-specific antibodies and flow cytometry. Unpublished data, Pharmingen R&D. 2.

Ulrike R. Schwarz, Jörg Geiger, Ulrich Walter, Martin Eigenthaler. 2003 Flow Cytometry Analysis of Intracellular Protein Phosphorylation for the Assessment of Activating and Inhibitory Signal Transduction Pathways in Human Platelets Thrombosis and Haemostasis. A preprint can be obtained, please contact M. Eigenthaler by email. 3.

van Huijsduijnen RH, Bombrun A, Swinnen D. 2002 Selecting protein tyrosine phosphatases as drug targets. Drug Discov Today 7(19):1013-9 4.

Wilhelm S, Chien DS. 2002 BAY 43-9006: preclinical data. Curr Pharm Des 8(25):2255-7. Bayer Research Center, Bayer Corporation 5.

Yoganathan N, Yee A, Zhang Z, Leung D, Yan J, Fazli L, Kojic DL, Costello PC, Jabali M, Dedhar S, Sanghera. 2002 Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties. J. Pharmacol Ther 93(2-3):233-42. 6.

Sue Chow, Harshna Patel, and David W. Hedley. 2001 Measurement of MAP Kinase Activation by Flow Cytometry Using Phospho-Specific Antibodies to MEK and ERK: Potential for Pharmacodynamic Monitoring of Signal Transduction Inhibitors. Cytometry (Communications in Clinical Cytometry) 46:72–78 7.

Demetri GD. 2001 Targeting c-kit mutations in solid tumors: scientific rationale and novel therapeutic options. Semin Oncol 28(5 Suppl 17):19-26 8.

Emery JG, Ohlstein EH, Jaye M. 2001 Therapeutic modulation of transcription factor activity. Trends Pharmacol Sci 22(5):233-40 9.

Wilkinson MG, Millar JB. 2000 Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J. Nov;14(14):2147-57

10. Yoganathan TN, Costello P, Chen X, Jabali M, Yan J, Leung D, Zhang Z, Yee A, Dedhar

蛋白质磷酸化与信号传导

S, Sanghera 2000 Integrin-linked kinase (ILK): a "hot" therapeutic target. J. Biochem Pharmacol Oct 15;60(8):1115-9

11. Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, Adams JL. 2000 Inhibition of

p38 MAP kinase as a therapeutic strategy. Immunopharmacology May;47(2-3):185-201 12. Ciardiello F, Caputo R, Bianco R, Damiano V, Pomatico G, De Placido S, Bianco AR,

Tortora G. 2000 Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor- selective tyrosine kinase inhibitor. Clin Cancer Res 6:2053-2063.

13. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE,

Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM. 1998 Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273:18623-18632.

蛋白质磷酸化与信号传导

您知道目前有多少螢光染劑可用於流式細胞儀嗎？

您知道誰是最大螢光標示抗體製造者嗎？

流式細胞儀自1980年代正式推出以來，不斷向更多色螢光分析、以及精確分選功能但操作簡便的目標挑戰，因此，新的機型不斷地被研發出。螢光染劑開發者也因應流式細胞儀的更新，不斷地生產出更多光穏定的螢光染劑，使得螢光染劑的選擇性大大地增加，不再只侷限於FITC、PE、以及PerCP等等傳統的螢光染劑。

BD Biosciences為辛苦的生醫研究人員提供最完整系列的螢光標示抗體，使您於有限的研究經費中可以進行最有效率的研究，能於最短時間內完成不可能的任務。您可以點選有興趣的螢光染劑，了解它們的特性、適用於那一機型的流式細胞儀、應使用那一個螢光參數接收訊號等等相關訊息，使您的實驗能更佳順利進行。

Alexa Fluor® 405

激發光源為405nm/407nm Solid State Violet雷射或是405nm Krypton雷射；其散發螢光與綠色螢光發生光譜重疊現象最低。 流式細胞儀種類 FACSVantage SE LSRII

雷射標示

Krypton 407nm (Laser 2)Violet 405nm (Laser 2) 或UV 355nm (Laser 2)

FACSAria

Violet 407nm (Laser 3)

偵測之螢光參數

FL4 B B B

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

蛋白质磷酸化与信号传导

Alexa Fluor® 488 與 Alexa Fluor®647

此兩種螢光染劑非常穏定，不論是偏酸性或偏醶性環境(pH4 – pH10)均不影響其螢光表現。

Alexa Fluor® 488

流式細胞儀種類 FACSCalibur/FACScan FACStar Plus FACSVantage SE LSR LSRII FACSAria

雷射標示

Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)

偵測之螢光參數

FL1 FL1 FL1 FL1 D E

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

Alexa Fluor®647

流式細胞儀種類 FACSCalibur 4 Color FACStar Plus FACSVantage SE LSR LSRII FACSAria FACSArray

雷射標示

Red Diode 635nm (Laser 2)HeNe 633nm (Laser 2) HeNe 633nm (Laser 2 / 3) HeNe 633nm (Laser 3) HeNe 633nm (Laser 4) HeNe 633nm (Laser 2) Red Diode 635nm (Laser 2)

偵測之螢光參數

FL4 FL3 FL4 FL6 (SSC-W)

B B B

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

Allophycocyanin (APC)

來自藍綠藻附光合色素，分子量約為105 kDa。屬於Phycobiliproteins家族。每個APC分子上有六個phycocyanobilin chromophores，與R-PE chromophore上的phycoerythrobilin結構相似。激發光源為635nm Red Diode雷射、以及633nm HeNe雷射。

流式細胞儀種類 FACSCalibur 4 Color FACStar Plus FACSVantage SE LSR LSRII FACSAria FACSArray

雷射標示

Red Diode 635nm (Laser 2)HeNe 633nm (Laser 2) HeNe 633nm (Laser 2 / 3)HeNe 633nm (Laser 3) HeNe 633nm (Laser 4) HeNe 633nm (Laser 2) Red Diode 635nm (Laser 2)

偵測之螢光參數

FL4 FL3 FL4 FL6 (SSC-W)

B B B

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以

蛋白质磷酸化与信号传导

APC- Cy7

結合APC與Cy7兩種螢光染劑之複合染劑。長時間曝露於可見光，螢光強度會減弱。散發螢光波長座落於遠紅光區(767nm)。若是使用紅光偵測接收器(例如Hamamatsu R3896光電倍增管)偵測APC-Cy7，建議使用750nm長通濾鏡。

下圖呈現APC-Cy7與APC光譜重疊情形，右圖則為經光補償調節後之圖形。

流式細胞儀種類 FACSCalibur 4 Color FACStar Plus FACSVantage SE LSR LSRII FACSAria FACSArray

雷射標示

Red Diode 635nm (Laser 2)HeNe 633nm (Laser 2) HeNe 633nm (Laser 2 / 3) HeNe 633nm (Laser 3) HeNe 633nm (Laser 4) HeNe 633nm (Laser 2) Red Diode 635nm (Laser 2)

偵測之螢光參數

FL4 FL4 FL5 FL6 (SSC-W)

A A A

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

Fluorescein isothiocyanate (FITC)

FITC分子量為389 Da，是最普遍用來製造抗體或是蛋白質試劑之螢光染劑，具有最高的量子產率(吸光能量轉換成散發螢光之效能)，大約一半的吸收光子均會散發螢光。由於FITC分子相當小，每一個抗體分子(或是蛋白質分子，或是Streptavidin，等等)通常與3~5

個FITC分子結合。

流式細胞儀種類 FACSCalibur/FACScan FACStar Plus FACSVantage SE LSR LSRII FACSAria

雷射標示

Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)

偵測之螢光參數

FL1 FL1 FL1 FL1 D E

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以

蛋白质磷酸化与信号传导

Pacific Blue®

使用UV光為激發光源，主要激發6,8-difluoro- 7- hydroxycoumarin fluorophore，即使於中性環境中也能呈現很強的螢光。 流式細胞儀種類 FACSVantage SE LSRII

雷射標示

Krypton 407nm (Laser 2)Violet 405nm (Laser 2) 或UV 355nm (Laser 2)

FACSAria

Violet 407nm (Laser 3)

偵測之螢光參數

FL4 B B B

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

R- phycoerythrin (R- PE)

螢光亮度最強之螢光染劑之一，來自紅藻的附光合色素，於紅藻細胞內擔任的光能轉運的角色，於光合作用中將光能轉送至葉綠體內。R-PE分子量為240 kDa，每個分子上帶有34個

phycoerythrobilin螢光分子，分子量較大，非常適合流式細胞應用，例如細胞表面抗原分子的絶對定量分析。

流式細胞儀種類 FACSCalibur/FACScan FACStar Plus FACSVantage SE LSR LSRII FACSAria FACSArray

雷射標示

Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Green Diode (Laser 1)

偵測之螢光參數

FL2 FL2 FL2 FL2 C D B

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

PE- Cy5 (正式名稱為 BD Cy- Chrome )

結合R- phycoerythrin (240- kDa蛋白質)及cyanine( Cy5，1.5 kDa)二種螢光染劑之複合染劑。由於這兩種螢光染劑間的光能轉換效率佳，使得吸收光的流失率低於5%，R-PE吸收大部份的光能後均能轉換為575nm螢光波長，再進而激發cyanine以產生更長波長的紅色螢光(785nm)。一般而言，每個抗體或是蛋白質平均結合一個PE-Cy5分子。

因為PE-Cy5複合染劑的激發波長範圍太廣，可被紅光雷射激發產生些許螢光，發生光譜重疊現象，所以不建議與633nm/635nm雷射激發之染劑(例如APC)合用。可與FITC與PE等螢光染劑合用。

由於cyanine易與細胞表面之Fc受體結合產生非特異性反應，所以使用此類抗體前，細胞必須先

蛋白质磷酸化与信号传导

與Fc Block作用以消除非特應性反應。

流式細胞儀種類 FACSCalibur/FACScan FACStar Plus FACSVantage SE LSR LSRII FACSAria

雷射標示

Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)

偵測之螢光參數

FL3 FL3 FL6 FL3 B C

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

PE- Cy7

為PE與Cy7之複合染劑，可與APC等(633nm/635nm激發光之)螢光染劑合用，光譜重疊現象較低。下圖為PE-Cy7與PE光譜重疊校正之圖形，左圖為未補償之圖形；右圖為經光補償校正，PE-Cy7干擾PE光區約佔2.7%。

流式細胞儀種類

FACSCalibur/FACScan FACStar Plus FACSVantage SE LSR LSRII FACSAria FACSArray

雷射標示

Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Green Diode (Laser 1)

偵測之螢光參數

FL3 FL3 FL3 FL3 A A A

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

蛋白质磷酸化与信号传导

PE- Texas Red®

為PE與Texas Red複合染劑，需要使用特別的光學濾鏡610/20nm。當與R-PE共同使用時，應特別小心光補償調節部份。

流式細胞儀種類 FACStar Plus FACSVantage SE LSR LSRII FACSAria

雷射標示

Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)

偵測之螢光參數

FL3 FL6 FL3 B C

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

Peridinin chlorophyll protein (PerCP)

為dinoflagellate (glenodinium)光合作用胞器的組成份子，為一35kDa蛋白質複合物。由於其光漂白特性，對於可見光非常敏感，螢光容易減弱，所以不建議使用於stream-in-air流式細胞儀。若是更換合適的光學濾鏡(例如675/22nm)，可提高螢光訊號接收的能力。

流式細胞儀種類 FACSCalibur/FACScan FACStar Plus FACSVantage SE LSR LSRII FACSAria

雷射標示

Argonm 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)Argon 488nm (Laser 1)

偵測之螢光參數

FL3 FL3 FL3 FL3 B C

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以確認螢光參數之位置。

PerCP- Cy5.5

為PerCP與Cy5.5之複合染劑，激發光源為488nm藍光雷射或是532nm綠光雷射。光敏性較PerCP低，螢光較持久。另外，與PE-Cy5相比較，較不易產生Fc受體致非特異性反應。

流式細胞儀種類 FACSCalibur/FACScan FACStar Plus FACSVantage SE LSR LSRII FACSAria FACSArray

雷射標示

Argon 488nm (Laser 1) Argon 488nm (Laser 1) Argon 488nm (Laser 1) Argon 488nm (Laser 1) Argon 488nm (Laser 1) Argon 488nm (Laser 1) Green Diode 532nm (Laser 1)

偵測之螢光參數

FL3 FL3 FL6 FL3 B B A

註：所列之流式細胞儀均為標準配備。若貴單位之儀器具有選配裝置，請與BD技術人員連絡以

蛋白质磷酸化与信号传导

確認螢光參數之位置。

Texas Red®

為sulforho-damine 101之is a sulfonyl chloride衍生物，分子量為625Da。Texas Red通常與advidin結合作為二次抗體以進行多色分析。一般常見的流式細胞儀由於激發光源與接收濾鏡波長範圍均不適合，所以無法使用。若是配備可調式激發波長之雷射(例如Krypton雷射)，將激發波長調至595-605nm範圍，則可與APC合用進行多色分析。

其他有關螢光染劑資料站

互動式螢光染劑光譜檢索站

BD流式細胞儀適用之常用螢光染劑全表 BD Biosciences常用螢光染劑光譜全表

BD Biosciences常用螢光染劑吸收光譜與散發光譜圖

New Fluorochrome Reagents for Immunology and Cell Signaling

Antibodies to Non-Human Primate Cell Surface Markers DESCRIPTION REACT CLONE

CD3

Bab, Cyno, Rhe

SP34-2

APPS FORMATFCM

APC-Cy7

SIZE CAT. NO. 100 tests

557757

FCM PE-Cy7 100 tests 557749 CD8

Bab, Cyno, Rhe

RPA-T8

FCM

APC-Cy7

100 tests

557760

FCM PE-Cy7 100 tests 557750

Antibodies to Human Cell Surface Markers DESCRIPTION REACT CLONE

CD3 CD4 CD8

CD11b/Mac-1

CD14 CD16

Hu

SK7

APPS FORMATFCM

PE-Cy7

SIZE CAT. NO. 100 tests

557851

FCM APC-Cy7100 tests 557832 Hu SK3 FCM PE-Cy7 100 tests 557852 RPA-T4Hu RPA-T8

FCM APC-Cy7

100 tests 557871

FCM PE-Cy7 100 tests 557746

100 tests 557834

SK1 FCM APC-Cy7Hu ICRF44

FCM PE-Cy7 100 tests 557743

FCM APC-Cy7100 tests 557754 Hu M5E2 FCM PE-Cy7 100 tests 557742

P9 FCM APC-Cy7

100 tests 557831 100 tests 557758

Hu 3G8 FCM APC-Cy7

FCM PE-Cy7 100 tests 557744

蛋白质磷酸化与信号传导

CD19 CD25 CD45 CD56 CD69

Isotype Controls for use with Antibodies to Human Cell Surface Markers DESCRIPTION REACT CLONEMouse IgG1

MOPC-21

APPS FORMAT

SIZE CAT. NO.

Hu SJ25C1

FCM PE-Cy7 100 tests 557835

FCM APC-Cy7100 tests 557791 Hu M-A251

FCM APC-Cy7

100 tests 557753

FCM PE-Cy7 100 tests 557741 Hu HI30 FCM PE-Cy7 100 tests 557748 2D1 FCM APC-Cy7

100 tests 557833

Hu B159 FCM PE-Cy7 100 tests 557747 Hu FN50 FCM APC-Cy7

100 tests 557756

FCM PE-Cy7 100 tests 557745

FCM PE-Cy7 100 tests 557872

FCM APC-Cy7100 tests 557873

Mouse IgG2a

Antibodies to Human Cytokines and Chemokine Receptors DESCRIPTION REACT CD195/CCR5

CCR7 IFN-γ IL-4 TNF-α

Hu

CLONE APPS2D7/CCR5

FCM

FORMAT APC-Cy7

SIZE CAT. NO. 100 tests

557755

G155-178

FCM APC-Cy7

100 tests 557751

FCMHu

3D12

FCMFCM

PE-Cy7 100 tests 557752 Alexa Fluor® 488100 tests Alexa Fluor® 647100 tests

557723 557734

FCMHu

B27

FCMFCM

PE-Cy7 100 tests 557648 Alexa Fluor® 488100 tests Alexa Fluor® 647100 tests

557718 557729

FCMHu Hu

MP4-25D2

MAb11

FCMFCMFCMFCM

PE-Cy7 100 tests 557643 Alexa Fluor® 488100 tests Alexa Fluor® 647100 tests Alexa Fluor® 488100 tests Alexa Fluor® 647100 tests

557727 557738 557722 557733

FCM

PE-Cy7 100 tests 557647

Isotype Controls for use with Antibodies to Human Cytokines and Chemokine Receptors

DESCRIPTION REACT CLONE Mouse IgG1

APPS FORMAT SIZE CAT. NO.

Alexa Fluor® 488 Alexa Fluor® 647

100 tests 100 tests

557721 557732

MOPC-21 FCM

FCM PE-Cy7 100 tests 557646

蛋白质磷酸化与信号传导

Antibodies to Mouse Cell Surface Markers

DESCRIPTION REACT CLONECD3 e (CD3　chain) CD4 (L3T4)

CD8a (Ly-2)

CD11b

(Integrin αm chain)

CD19

CD25 (IL-2R α chain)

CD45 (LCA, Ly-5)

Antibodies to Mouse Cell Surface Markers (continued) DESCRIPTION REACT CLONECD45R/B220

Ms

RA3-6B2

APPS FORMATFCM

APC-Cy7

SIZE CAT. NO. 0.1 mg

552094

FCM PE-Cy7 0.1 mg 552850 Ms 1D3 FCM APC-Cy7

0.1 mg 557655

Ms

145-2C11

APPS FORMATFCM

APC-Cy7

SIZE CAT. NO. 0.1 mg

557596

FCM PE-Cy7 0.1 mg 552774 Ms GK1.5 FCM APC-Cy7

RM4-5

FC M

PE-Cy7

0.1 mg 552051 0.1 mg

552775

Ms 53-6.7 FCM APC-Cy70.1 mg 557654

FCM PE-Cy7 0.1 mg 552877 M1/70 FCM APC-Cy7

0.1 mg 557657

FCM PE-Cy7 0.1 mg 552854 Ms PC61 FCM APC-Cy7

0.1 mg 557658

FCM PE-Cy7 0.1 mg 552880 Ms 30-F11

FCM APC-Cy7

0.1 mg 557659

FCM PE-Cy7 0.1 mg 552848

FCM PE-Cy7 0.1 mg 552772 FCM PE-Texas 0.1 mg

Red

551489

CD49b (Integrin 2

chain) CD69 (Very Early Activation Ag) CD95 (Fas)

IgM

Ly-6G and Ly-6C

(Gr-1)

NK-1.1 (NKR-P1B &

NKR-P1C)

H　2 FCM APC-Cy70.1 mg 557656

H1.2F3FCM PE-Cy7 0.1 mg 552879

Ms Jo2 FCM PE-Cy7 0.1 mg 557653 Ms R6-60.2Ms RB6-8C5

FCM PE-Cy7 0.1 mg 552867 FCM PE-Cy7 0.1 mg 552985

PK136FCM PE-Cy7 0.1 mg 552878

Isotype Controls for use with Antibodies to Mouse Cell Surface Markers DESCRIPTION REACT CLONE

APPS FORMAT

SIZE CAT. NO.

蛋白质磷酸化与信号传导

Hamster IgG1 (anti-TNP)

Hamster IgG2

Rat IgG1 (anti-KLH)

Rat IgG2a

Rat IgG2b

Mouse IgG2a

Antibodies to Mouse Cytokines DESCRIPTION REACT CLONE

IFN-γ IL-2 IL-4 TNF-α

Isotype Controls for use with Antibodie s to Mouse Cytokines DESCRIPTION REACT CLONERat IgG1

Rat IgG2b

BDTM PhosFlow Antibody Reagents DESCRIPTION REACT CLONECaveolin1 (pY14) Hu, Ms, Rat

56

APPSFCM

FORMAT SIZE CAT. NO.PE

50 tests

612568

R3-34

APPSFCM

FORMAT Alexa Fluor® 488Alexa Fluor® 647

SIZE CAT. NO. 0.1 mg 0.1 mg 0.1 mg 0.1 mg

557720 557731

Ms

XMG1.2

APPSFCM FCM

FORMAT Alexa Fluor® 488 Alexa Fluor® 647

SIZE 0.1 mg 0.1 mg

CAT. NO. 557724 557735

FCM Ha4/8 FCM FCM A110-1

FCM FCM

FCM R35-95

FCM A95-1 FCM FCM G155-178

FCM

PE-Cy7 0.1 mg 552811 APC-Cy7

0.1 mg 0.1 mg 0.1 mg 0.1 mg

557664 557652 557663 552869 552770 552784 552773 552849 552868

PE-Cy7 0.1 mg APC-Cy7

PE-Cy7 0.1 mg APC-Cy7

PE-Cy7 0.1 mg APC-Cy7

PE-Cy7 0.1 mg PE-Cy7 0.1 mg

A19-3

FCM

AP C-Cy7

0.1 mg

557662

FCM PE-Cy7 0.1 mg 557649 Ms Ms Ms

JES6-5H4

11B11 MP6-XT22

FCM FCM FCM FCM FCM FCM

Alexa Fluor® 488 Alexa Fluor® 647 Alexa Fluor® 488 Alexa Fluor® 647 Alexa Fluor® 488 Alexa Fluor® 647

0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg 0.1 mg

557725 557736 557728 557739 557719 557730

FCM PE-Cy7 0.1 mg 557644

FCM FCM A95-1 FCM FCM FCM

PE-Cy7 0.1 mg 557645 Alexa Fluor® 488Alexa Fluor® 647

557726 557737

PE-Cy7 0.1 mg 557651

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