在这里, 提出了一种中-高通量分析在细胞级蛋白质磷酸化事件的协议。磷流式细胞术是表征信号畸变、识别和验证生物标志物、评估药效学的有力手段。
异常细胞信号在肿瘤的发展和进展中起着重要的作用。大多数新颖的靶向疗法确实是针对蛋白质和蛋白质功能, 细胞信号畸变可能因此作为标志物, 以表明个性化的治疗方案。与 DNA 和 RNA 分析相反, 蛋白质活动的变化可以更有效地评估药物敏感性和耐药性的机制。磷流式细胞术是一种功能强大的技术, 用于测量细胞水平的蛋白质磷酸化事件, 这是区别于其他基于抗体的方法的一个重要特征。该方法允许同时分析多种信号蛋白。结合荧光细胞条形码, 在短时间内, 标准细胞仪硬件可以获得较大的中、高吞吐量数据集。磷流式细胞术在基础生物学研究和临床研究中都有应用, 包括信号分析、生物标志物发现和药效学评价。以慢性淋巴细胞白血病细胞为例, 为纯化外周血单个核细胞磷流分析提供了详细的实验协议。
磷流式细胞仪用于分析单细胞分辨率下蛋白质磷酸化水平。该方法的总体目标是在特定条件下映射细胞信号模式。利用流式细胞仪的多参数容量, 可以同时在不同的非均匀细胞群 (如外周血) 中同时分析几种信号通路。这些特性比其他基于抗体的技术具有优势, 如免疫组化、酶联免疫吸附试验 (ELISA)、蛋白质阵列和反相蛋白阵列 (RPPA)1。磷流式细胞术可与荧光细胞条形码 (FCB) 相结合, 这意味着单个细胞样本被标记为具有独特的荧光染料特征, 使它们可以混合在一起, 染色和分析为单个样品2。这降低了抗体的消耗, 提高了数据的鲁棒性, 通过组合控制和处理样本, 并提高了获取速度。组合 FCB 人口可以被分成较小的样本和染色多达35种不同的磷特定的抗体, 取决于数量的起始材料。因此, 大型分析实验可以使用标准的细胞仪硬件运行。磷流式细胞术应用于多例血液肿瘤患者样本中的特征信号通路, 包括慢性淋巴细胞白血病3、4、5、急性髓系白血病 (AML)6和非霍奇金淋巴瘤7。磷流式细胞术是一种有效的方法来表征信号畸变, 识别和验证生物标志物, 并评估药效学。
给出了磷流式细胞仪分析慢性淋巴细胞白血病患者标本的优化方案 (图 1A)。以基底信号特征、抗 IgM/B 细胞受体刺激和药物摄动为例。提供了一个 FCB 矩阵的详细描述。该协议可以很容易地适应其他悬浮细胞类型。
磷流式细胞术是测定单细胞蛋白质磷酸化水平的一种强有力的技术。由于该方法依赖于抗体的染色, 磷流式细胞术受抗体可用性的限制。此外, 为了获得可靠的结果, 所有抗体应在使用前滴定和验证。磷特定抗体的滴定详细的协议在别处被描述了12。在面板设计中, 考虑信噪比是至关重要的。在这个例子中, 所有的磷抗体被共轭到 Alexa 647。这种荧光通常提供低与高含量的磷蛋白</…
The authors have nothing to disclose.
这项工作是在谢蒂尔·保尔森 Taskén 教授实验室进行的, 并得到了挪威癌症协会和 Stiftelsen 克里斯蒂安·卡尔森捷成的支持。约翰. Landskron 和玛丽安客运被承认对手稿的批判性阅读。
RPMI 1640 GlutaMAX | ThermoFisher Scientific | 61870-010 | Cell culture medium |
Fetal bovine serum | ThermoFisher Scientific | 10270169 | Additive to cell culture medium |
Sodium pyruvate | ThermoFisher Scientific | 11360-039 | Additive to cell culture medium |
MEM non-essential amino acids | ThermoFisher Scientific | 11140-035 | Additive to cell culture medium |
Lymphoprep | Alere Technologies AS | 1114547 | Density gradient medium |
Anti-IgM | Southern Biotech | 2022-01 | For stimulation of the B cell receptor |
BD Phosflow Fix Buffer I | BD | 557870 | Fixation buffer |
BD Phosflow Perm Buffer III | BD | 558050 | Permeabilization buffer |
Alexa Fluor 488 5-TFP | ThermoFisher Scientific | A30005 | Barcoding reagent |
Pacific Blue Succinimidyl Ester | ThermoFisher Scientific | P10163 | Barcoding reagent |
Pacific Orange Succinimidyl Ester, Triethylammonium Salt | ThermoFisher Scientific | P30253 | Barcoding reagent |
Compensation beads | Defined by user | Correct species reactivity | |
Falcon tubes | Defined by user | ||
Eppendorf tubes | Defined by user | ||
96 well V-bottom plates | Defined by user | Compatible with the flow cytometer | |
Centrifuges | Defined by user | For Eppendorf tubes, Falcon tubes and plates | |
Water bath | Defined by user | Temperature regulated | |
Flow cytometer | Defined by user | With High Throughput Sampler (HTS) | |
Name | Company | Catalog Number | Comments |
Antigen | |||
AKT (pS473) | Cell Signaling Technologies | 4075 | Clone: D9E Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 |
ATF-2 (pT71) | Santa Cruz Biotechnology | sc-8398 | Clone: F-1 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
BLNK (pY84) | Beckton Dickinson Pharmingen | 558443 | Clone: J117-1278 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
Btk (pY223)/Itk (pY180) | Beckton Dickinson Pharmingen | 564846 | Clone: N35-86 Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
Btk (pY551) | Beckton Dickinson Pharmingen | 558129 | Clone: 24a/BTK (Y551) Reference: Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 |
Btk (pY551)/Itk (pY511) | Beckton Dickinson Pharmingen | 558134 | Clone: 24a/BTK (Y551) Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 |
CD3ζ (pY142) | Beckton Dickinson Pharmingen | 558489 | Clone: K25-407.69 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
Histone H3 (pS10) | Cell Signaling Technologies | 9716 | Clone: D2C8 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
IκBα | Cell Signaling Technologies | 5743 | Clone: L35A5 Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
LAT (pY171) | Beckton Dickinson Pharmingen | 558518 | Clone: I58-1169 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
Lck (pY505) | Beckton Dickinson Pharmingen | 558577 | Clone: 4/LCK-Y505 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
MEK1 (pS298) | Beckton Dickinson Pharmingen | 560043 | Clone: J114-64 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
NF-κB p65 (pS529) | Beckton Dickinson Pharmingen | 558422 | Clone: K10-895.12.50 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
NF-κB p65 (pS536) | Cell Signaling Technologies | 4887 | Clone: 93H1 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 |
p38 MAPK (pT180/Y182) | Cell Signaling Technologies | 4552 | Clone: 28B10 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
p44/42 MAPK (pT202/Y204) | Cell Signaling Technologies | 4375 | Clone: E10 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
p53 (pS15) | Cell Signaling Technologies | NN | Clone: 16G8 Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
p53 (pS20) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
p53 (pS37) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
p53 (pS46) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
p53 (pS392) | Cell Signaling Technologies | NN | Clone: Polyclonal Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96 |
PLCγ2 (pY759) | Beckton Dickinson Pharmingen | 558498 | Clone: K86-689.37 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
Rb (pS807/pS811) | Beckton Dickinson Pharmingen | 558590 | Clone: J112-906 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
S6-Ribos. Prot. (pS235/236) | Cell Signaling Technologies | 4851 | Clone: D57.2.2E Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
SAPK/JNK (pT183/Y185) | Cell Signaling Technologies | 9257 | Clone: G9 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55 |
SLP76 (pY128) | Beckton Dickinson Pharmingen | 558438 | Clone: J141-668.36.58 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 |
STAT1 (pY701) | Beckton Dickinson Pharmingen | 612597 | Clone: 4a Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
STAT3 (pY705) | Beckton Dickinson Pharmingen | 557815 | Clone: 4/P-STAT3 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
STAT4 (pY693) | Zymed/ThermoFisher Scientific | 71-7900 | Clone: Polyclonal Reference: Uzel et al., 2001, Detection of intracellular phosphorylated STAT-4 by flow cytometry, Clin Immunol, 100(3): 270-6 |
STAT5 (pY694) | Beckton Dickinson Pharmingen | 612599 | Clone: 47/Stat5(pY694) Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |
STAT6 (pY641) | Beckton Dickinson Pharmingen | 612601 | Clone: 18/P-Stat6 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 |
SYK (pY525/Y526) | Cell Signaling Technologies | 12081 | Clone: C87C1 Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62 |
ZAP70/SYK (pY319/Y352) | Beckton Dickinson Pharmingen | 557817 | Clone: 17A/P-ZAP70 Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410 Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9 Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770 |