pROTOCOLS - 抗体・試薬・ELISAのeBioscience
Contents
1. eFluor 2 2 eFluor 222. N 350 325 APC 300 B PE 225 PE Cy7 Alexa Fluor 700 200 eFluor 650 175 PerCP Cy5 5 Alexa Fluor 647 EE 125 APC eFluor 780 eFluor 450 0 100 eFluor 605 75 FITC y 0 Alexa Fluor 488 E 4 5 7 dii 0 7 7 1 CD4 eBioscience LSRII FLOW CYTOMETRY Section Ill Spectral Overlap amp Compensation MULTICOLOR FLOW CYTOMETRY
3. 1 1 Fluorescence Minus One FMO 1 F M O _eFluor _eFluor E APC Alexa _eFluor Alexa
4. 1 a b 2 2 Excitation Laser nm Fluorochrome eFluor 450 Pacific Blue 405 407 450 405 407 455 Emission maximum nm eFluor 605 eFluor 625 C eFluor 650 355 405 407 355 405 407 355 405 407 605 625 650 FITC 488 518 Alexa Fluor 488 488 519 PE 488 575 PE Cy5 488 667 PE Cy5 5 488 695 PerCP Cy5 5 488 695 PE Cy7 488 785 APC 633 635 640 660 Alexa Fluor 647 633 635 640 668 Alexa Fluor 700 633 6
5. PS PS CEES Ca2 V PS PS V PS DNA DNA KAD NA 50 200kb 180 2
6. Phenotype Acute Myeloid Leukemia AML B Acute Lymphoblastic Leukemia B ALL T Acute Lymphoblastic Leukemia T ALL Myeloma Breast Medullo blastoma Glioblastoma Head amp Neck Sqamous Cell Carcinoma Colon Melanoma Prostate Pancreas Lung non small cell Hepatocellular Cancer Stem Cell Phenotypes CD34 CD38 CD34 CD10CD19 CD34 CD7 or CD34 CD4 CD138CD34 CD44 CD2479w CD133 CD133 CD44 EpCAMPCD44 CD166 or CD133 ABCB5 CD44 CD44 CD24 EpCAM or CD133 CDT35 CD44 CD90 eBioscience offers an extensive selection of antibody reagents cytokines and growth factors relevant to all areas of stem cell research including iPS cell ES cell hematopoietic stem Bioscience MPacific cell and cancer stem cell Blue Cat research No 88 7773 APC c Kit cat 17 1171 PE Sca 1 cat 12 5981 SD 224
7. MHC MHC CD1a CD11c CD11b CD4 CD85g ILT7 CD85k ILT3 Langerin CD123 x x x x x Human Mouse Dendritic Cell Biomarkers Expressed OSubset Not Expressed ITIC CELL Identification of human and mouse plasmacytoid dendritic cells Top panel Staining of normal human peripheral blood cells with PE anti human CD123 Cat No 12 1239 and Alexa Fluor 647 anti human ILT7 Cat No 51 5179 to identify the pDC population Bottom panel Staining of mouse splenocytes with FITC anti mouse B220 Cat No 11 0452 and Alexa Fluor 647 anti mouse siglec H Cat No 51 0333 to identify the pDC population CD8a CD205 B220 Langerin E Cadherin Siglec H xix x xi xix x ox lt o o CD205 PE Cy7 CD205 DEC205 expression on mouse dendritic cell subsets Mouse splenocytes were prepared as a single cell suspension and stained with PE 7 anti mouse CD205 Cat No 25 2051 FITC anti mouse CD11c Cat No 11 0114 and APC anti mouse CD8a
8. 2 Band Pass Filter 440 40 450 50 440 40 450 50 605 40 605 50 660 40 530 30 530 30 575 26 585 40 670 14 695 40 695 40 695 40 710 40 780 60 780 40 660 20 660 20 710 40 710 50 720 45 780 60 780 60 _Dichroic Pass Filter 685 LP 735 LP 735 LP NOTE 1 eBioscience stain index 2 Meang Meang
9. 145 899 SOOM eS 2 PE CD4 PE
10. 60 1 1 2x106cells ml PBS 1 w v 2 30 3 300 400xg 4 5 4 PBS 2 300 400xXg 4 5 5 6 1 2X106cells ml 70 v v 30 70 v v 20 12 18 70 v v 20
11. 14 1ml Rinse Buffer red cap 2 300 400Xg 4 5 15 0 1ml 16 IC PRB 1 30 17 0 9ml Propidium lodide RNase Solution amber bottle 0 1ml NOTE PI RNase A solution 0 5ml 18 30 19 Propidium lodide RNase Solution 20 3 FLOW CYTOMETRY FSC AND SSC PROFILES MOUSE SPLEEN
12. mitochondrial outer membrane permeabilization MOMP C Apaf 1 Fas death AE caspase caspase pro enzyme caspase Apoptosis DNA AO RES caspase CHOC
13. 3 2 Accutase W Enzyme Cell Detachment Medium 05786 3 NOTE Accutase 3 2 107ml Flow Cytometry Staining Buffer I B 60mmX15mm 3ml eBioscience Flow Cytometry Staining Buffer eBioscience Cat No 00 4222 15 50ml
14. eBioscience has an extensive selection of reagents to study all aspects of B cell biology including their development function and pathogenesis KEY PRODUCTS AID CD20 CD66a FREB ENTIAL RODUCTS BP 1 CD19 CD45R B220 CD43 CD93 AA4 1 ESS p BB SLE RA References Hardy RR Kincade PW Dorshkind K The protean nature of cells in the B lymphocyte lineage Immunity 2007 Jun 26 6 703 14 Review LeBien TW Tedder TF B lymphocytes how they develop and function Blood 2008 Sep 1 112 5 1570 80 Review FLOW CYTOMETRY Treg REGULATING IMMUNE RESPONSES _ regulatory TAHAE Treg Treg Treg Treg
15. 3 hydroxyI DNAR BrdU Apo BrdU Apoptosis Detection Kit eBioscience Cat No 88 6671 12X75mm PBS 1 w v 74 LABELING SOLUTION 1ASSAY 5ASSAYS TdT Reaction Buffer green 10 00ul 50 00 ul cap 10 ASSAYS 100 00 ul 0 75 ul 8 00 ul 32 25 ul 51 00 pl 3 75 ul 40 00 ul 161 25 ul 255 00 ul 7 50 yl 80 00 ul 322 50 ul 510 00 ul TdT Enzyme yellow cap Br dUTP violet cap Distilled H O Total Volume DNA Labeling Solution NOTE 1 DNA DNA DNA 24 ANTIBODY SOLUTION Flourescein PRB 1 orange cap ASSAY 10 ASSAYS 25 00 ul 475 00 ul 500 00 pl Rinse Buffer red cap Total Volume Antibody Solution Apo BrdUTM Kit
16. NK NK MHC Class NK NK NK perforin granzyme B NK Ly49 KIR CD49 NKG2A NK NKG2D NK
17. Treg CD39 CD73 CTLA 4 Foxp3 Treg in vitro in vivo ANTIGEN HUMAN MOUSE CD4 CD25 CD39 CD62L CD73 CD101 CD103 CD127 CD134 CD152 CTLA 4 CD223 LAG 3 00 0000 Foxp3 FR4 GITR AITR IL 10 TGF 3 Reported phenotypic profile of Treg cells Pan Marker Subset w Down regulated eBioscience is the industry leader in providing reagents to analyze Tregs including Foxp3 antibodies and new antibodies for sorting of highly purified viable Tregs KEY PRODUCTS CD39 CD73 CD39PE Cy7 CD101 FR4 Human Foxp3 Whole Blood Staining Kit 96 of Max ESSENTIAL PRODUCTS CD4 CD25 CD127 a 9 Foxp3 FR4 FITC AITR GITR 6010 PE CD73 eFluor 450 0 References Treg Fernandez l et al 2007 CD101 surface expression discriminates CD101
18. Treg Phenotype Foxp3 transcription factor Treg T Treg GITR CTLA 4 CD39 CD73 CD101 CD103 CD127 FR4 Treg Treg ecto enzyme CD39 CD73 ATP CD39 CD73 T A2A
19. 000 0105109120 CAEL HSC Counts o Nanog Alexa Fluor 647 2006 Oct3 4 Sox2 c Myc KIf4 iPS 2007
20. NK Feature Phenotype TCR rearrangement CD1d restricted a GalCer reactive Surface expression and functional differences between NK and NKT cells IFN g NK NK T B NK T CD3TCRCD94 NKp46 NKT Va24Ja18T FITC CD3 Cat 11 0038 PE IgG1 Cat N
21. 515nm MEEI 635nm 2 3 BrcdUTP DNA BrdU DNA OD RNaseA DNA Early Apoptosis Late Apoptosis Non apoptotic Cells Annexin V FITC Apoptosis Detection Kit Mouse thymocytes were prepared as a single cell suspension and incubated overnight at 37 C in medium left panel or medium with 1 uM dexamethasone right panel Cells were harvested and stained using the Annexin V FITC Apoptosis Detection Kit Cat No 88 8005 and Propidium lodide Staining Solution Cat No 00 6990 95 TUNEL
22. 4 2 3 Protocol on ice 4 4 eFluor W nanocrystal NC 5 BSA FCS www drmr com compensation index html www cyto purdue edu index htm I A A1 Bel 775 12X75mm 96 well 2 Affinity CD16 32 eBioscience Cat No 14 0161 Affnity
23. T Conventional DC CD34 Toll
24. potency among murine FoxP3 regulatory T cells J Immunol CD101 7928082814 2 2 Deaglio S et al 2007 Adenosine generation catalyzed by CD39 and Treg CD73 expressed on regulatory T cells mediates immune suppression J Exp Med 204 257 1265 Wing K et al 2008 CTLA 4 control over Foxp3 regulatory T cell function Science 322 271 275 FLOW CYTOMETRY APOPTOSIS PROGRAMMED CELL DEATH Apoptosis Apoptosis 3 induction activation execution 3 Apoptosis UV DNA
25. 5 4C on ice 20 30 NOTE 6 Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer 2ml 1well 200l 4 5 300 400xg 2 7 50001 Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer 8 100ul Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer 39072 4 15 30
26. eBioscience Fixation Permeabilization Kit eBioscience Cat No 88 8823 10X Permeabilization Buffer LG 1 2 LT 3 4ml Permeabilization Buffer Ela in vivo 14 2 Section Cell Preparation Protocol A 1 II A2 100uI IC Fixation Buffer 20 6 1ml 1xPermeabilization 13 14 15 Buffer 300 400xg 4 5
27. DNA References Darzynkiewicz Z Huang X and Okafuji M 2006 Detection of DNA strand breaks by flow and laser scanning cyometry in studies of apoptosis and cell proliferation DNA replication Methods Mol Biol 314 81 93 eBioscience has a variety of tools to allow the phenotyping and characterization of cells undergoing apoptosis KEY PRODUCTS Annexin V Apoptosis Detection Kit FITC APC APO BrDU Apoptosis Detection Kit APO DIRECT Apoptosis Detection Kit Propidium lodide Nur77 ESSENTIAL PRODUCTS Bcl 2 Cytochrome C CD95 Fas CD178 FasL TRAIL FLOW CYTOMETRY NK T CELL BRIDGING INNATE amp ADAPTIVE IMMUNITY NK NK missing self
28. Pax5 B CD19 eae 7c B fed Behe cts CD21 CD23 CD24 AA4 1 B ALEF Activation induced cytidine deaminase AID Uncommitted CD45R B220 CD19 CD43 CD24 HSA CD93 AA4 1 1 Surface IgM Surface IgD CD2 CD22 CD21 CD23 CD79a CD79b 19 a B CD127 IL 7Ra c Kit CD117 Phenotype of B cells during progressive stages of maturation from uncommitted progenitor to mature B cell B cell phenotyping Mouse splenocytes were stained with the following panel of antibodies PerCP Cy5 5 anti mouse CD19 Cat No 45 0193 Pacific Blue anti mouse Gr 1 Cat No 57 5931 Pacific Blue anti mouse CD11b Cat No 57 0112 PE anti mouse CD23 Cat No 12 0232 eFluor 60556 anti mouse CD24 Cat No 93 0242 FITC anti mouse IgD Cat No 11 5993 PE Cy7 anti mouse IgM Cat No 25 5790 and APC anti mouse CD93 Cat No 17 5892 Following exclusion of Gr 1 and
29. a KERA 2 B Cells CD44 APC 9 403 102 105 CD62L eFluor 60556 Cells 9 102 104 10 NK1 1 PE NK Cells CD44 APC 9 403 104 105 CD62L eFluor 60556 Multicolor phenotypic analysis of mouse splenocytes Mouse splenocytes were stained with the following panel of antibodies eFluor 650 anti mouse B220 coming soon PE Cy7 anti mouse CD3 Cat No 25 0031 eFluor 450 anti mouse CD4 Cat No 48 0042 APC eFluor 780 anti mouse CD8 Cat No 47 0081 PE anti mouse NK1 1 Cat No 12 5941 APC anti mouse CD44 Cat No 17 0441 and eFluor 60550 anti mouse CD62L Cat No 93 0621 The contour plots show the initial gating to discriminate the B220 B cells green and CD3 T cells gray The CD3 cells are further subset into CD4 turquoise and CD8 pink populations The non B or T cell B220 CD3 population contains the NK1 1 NK subset blue histogram expression of CD44 and CD62L was evaluated on each of the lymphocyte subpopulations CD44 APC CD8 APC eFluor 780
30. 3 2ml PBS 4 300 400Xg 4 5 5 2ml 1XBinding Buffer 6 300 400xg 4 5 7 1 5X106 ml 1XBinding Buffer CEA 8 5ul Annexin V 100ul 9 10 15 10 2ml 1XBinding Buffer 300 400Xg 5 1 200u 001xBinding Buffer CBE Ag Lx 3 12 5 1 Propidium lodide Staining Solution 13 PI PE 51 59 14
31. DL 100ml 1XPermeabilization Buffer 300 400Xqg 4 5 100ul 1XPermeabilization Buffer 20 1ml 1XPermeabilization Buffer 300 400xg 4 5 595 1ml Flow Cytometry Staining Buffer 300 400Xg 4 5 500ul Flow Cytometry Staining Buffer I B2 12X75mm eBioscience Foxp3 Staining Buffer Set eBioscience Cat No 00 5523 eBioscience Flow Cytometry Staining Buffer eBioscience Cat No 00 4222 Normal Rat Serum eBioscience Cat No 24 5555 Normal Mouse Serum eBioscienc
32. 500ul Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer Ant quiatur Vendita tendit pedit ipis secea cusa volecto reprepe rchiti comnitaque lam nus ut dolupta tempost FLOW CYTOMETRY Section Il Immunofluorescent Staining II B I B1 2 in vitro in vivo eld in vitro in vitro 4 6 eBioscienceTM Monensin Solution eBioscience Cat No 000 4505 eBioscienceTM Brefelcin Solution eBioscience Cat No 000 4506 00 4 27x 12x75mm
33. 2X 1212 1 eBioscience CD4
34. LES 2m 1well 200ul 300 400Xg 4 5 HAERERE 8 500 1 Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer 9 100ul Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer 4 15 30 10 Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer 2ml 1well 200 4 5 300 400xg 2 11
35. Cat No 88 8823 eBioscience FoxP3 Cat No 00 5523 APO CD45 64K 128K 192K 256K CD45 LCA eBioscience Foxp3 Staining Buffer Set 64K 128K 192K 256K 0 02 104 10 Figure 1 Fixation and Permeabilization HUMAN LWB Lysed Whole Blood HUMAN PBMC Peripheral Blood Mononuclear Cells 128K SSC 8 64K 28K 19100 250K 101 102 103 104 10 64K FoC 128K 192K 256K 101 102 103 104 10 CD45 FITC FSC CD45
36. Jc UR Rer BIO SEE FSC SSC eem rJ ae Fixation amp Permeabilization Kit 1 eBioscience
37. 1 100ul well 2 20 20ul Affinity FcyR 3 50ul Flow Cytometry Staining Burer eFluor NC Flow Cytometry Staining Buffer 4 4 20 30 NOTE D 5 1well 1XRBC Lysis Buffer 2ml 10 RBC Lysis Buffer 15 6 300 400Xg 4 5 7 Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer
38. 9 Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer 2ml 1well 200Al 4 5 300 400Xg 2 10 500ul Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer Ca 2 eBioscience TM 1XRBC Lysis Buffer eBioscience Cat No 00 4333 12X75mm 96 well pup pe 2 i uds FcyR eBioscience Cat No 14 9161 eBioscience Flow Cytometry Staining Buffer eBioscience Cat No 00 4222 eFluor NC Flow Cytometry Staining Buffer eBioscience Cat No 00 3222 NOTE eFluorTM nanocrystal NC
39. 1 277 VET 3ml 10ml Flow Cytometry Staining Buffer 2 2 10mL Flow Cytometry Staining Buffer 3 4 5 300 400Xg 4 4 5 Step3 2x107mll Flow Cytometry Staining BufferCH Ral 3 I C e Scissors Or scalpel blade PBS or other suitable buffer 60mmx15mm eBioscience Flow Cytometry Staining Buffer eBioscience Cat No 00 4222 15
40. 50ml 1 2 4mm 2 PBS NOTE wwwrtissuedissociation Com 3 4 4 5 300 400xg 4 5 PBS 6 Step4 4 7 Step5 6 8 Flow Cytometry Staining Buffer
41. PRODUCTS CD34 CD133 Nanog Oct 3 4 SSEA c Kit APC ESSENTIAL PRODUCTS CD90 CD117 CD135 CXCR4 Sca 1 Sca 1 PE CSC iPS AL S iPS T oe A A A Ci Ose om memes Takahashi et al 2007 Induction of pluripotent stem cells from ad
42. FcyR eBioscience Cat No 14 9161 eBioscienceTM Flow Cytometry Staining Buffer eBioscience Cat No 00 4222 eFluor NC Flow Cytometry Staining Buffer eBioscience Cat No 00 3222 1 Section 1 eFluorTM nanocrystal NC 2X107 ml eFluorTMNC Flow Cytometry Staining Buffer mea UCC IE EL 2 Fc 20 106 0 5 1ug CD16 CD32 20 106 20ul Affnity FcyR 3 50 06 well 4 100u ie 50ul 50ul Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer
43. PE 488nm FITC PerCP Cy5 5 PE Cy5 5 PE 405nm TERETA ui eFluor605 PE CD4 PE
44. T T Th1 Th2 in vitro in vivo IL 17 T Th17 Th17 Th17 Th17 ROR y t RORy t RORy Th17 RORYt IL 17 Th17 Feature Surface Expression IL 12Rb2 IFN y R Tim 3 IL 4R IL 17R 8 Cytokine Expression IFN y IL 4 IL 5 IL 13 T bet Gata 3 Master Regulator Transcription Factor STAT Regulators Supportive Cytokines
45. Classical NKT Karre K Ljunggren HG Piontek G Kiessling R Selective rejection Type INKT Va14Ja18 Va24Ja18 of H 2 deficient lymphoma variants suggests alternative immune invariant T invariant iNKT defence strategy Nature 1986 Feb 20 26 319 6055 675 8 CD160 CD1d NKT Bendelac A Killeen N Littman DR Schwartz RH A subset of CD4 T typell thymocytes selected by MHC class molecules Science 1994 Mar 25 263 5154 1774 8 Vivier E Tomasello E Baratin Walzer Ugolini S Functions of natural killer cells Nat Immunol 2008 9 5 503 10 Review NKT FLOW CYTOMET F DIRECTING AN IMMUNE RESPONSE DC T T
46. PE 2 Fluorochrome in Detecto FITC PE PerCP Cy5 5 PE Cy7 APC Alexa Fluor 700 APC Alexa Fluor 750 eFluor 450 eFluor 605 eFluor 650 2 CD4
47. viability dye Annexin V positive 7 AAD PI negative Annexin V 7 AAD PI double positive ow V FITC Apoptosis Detection Kit eBioscience Cat No 88 8005 or Annexin V APC Apoptosis Detection Kit eBioscience Cat No 88 8007 Propidium lodide Staining Solution eBioscience Cat No 00 6990 PBS 10Xstock 1X Binding Buffer NOTE Annexin V PS Annexin V EDTIA inner leaflet Annexin VO Annnexin V Annnexin V Binding Buffer 1 2 300 400xg 4 5
48. Annexin V PI FLOW CYTOMETRY Section Il Immunofluorescent Staining 1 4 Apo BrdU Kit for detection of late stage apoptosis DNA DNA 3 hydroxyl terminal deoxynucleotidyl transferase TdT lk DNA 3 hydrokyl nucleoside analog bromodeoxyuridine triphosphate BrdU BrdU APO BrdUTM Kit DNA
49. 9 Step4 2X107 ml Flow Cytometry Staining Buffer I D PBMC PBS Ficoll or other density separation medium eBioscienceTM Flow Cytometry Staining Buffer eBioscience Cat No 00 4222 15 50ml 1 50ml PBS 1 1 ROUE d 2 Ficoll O 3 20 700Xg 4 PBMC 5 PBS 6 4 4 5 300 400Xo 7 Flow Cytometry Staining Buffer 8 Step6 2X107ml Flow Cytometry Staining BufferCH Ral 9 FLOW CYTOMETRY Section Il Immunofluorescent Staining General Notes 1
50. STAT 1 4 IL 12 IL 27 IFN y STAT 6 IL 25 IL 17E Properties of helper T cell lineages Th17 Th17 Th1 Th2 Th1 Th2 IFN y PIL 4 TGF IL 6 Th17 TNF IL 1 IL 6 IL 21 IL 4 TGF B Identification of Th17 cells by flow cytometric detection of ROR y t CD4 T cells were sorted from RORy t deficient top dot plot or wildtype bottom dot plot mouse spleen and lymph node cultured in Th17 polarizing conditions for 3 days and stained with anti mouse CD4 PE Cy5 5 anti mouse IL 17A APC and anti mouse human ROR y t PE Cat No 12 6988 The histogram shows staining of RORy t in CD4 IL 17A events from RORy t deficient mice blue line and wildtype mice pink line Cells in the lymphocyte gate were used for analysis Data provided courtesy of D R Littman Laboratory RORyt RORyt IL 1R1 IL 23R CCR6 h CD161 h CD25 CD39 CD73 CD101 CD127 FR4 GITR IL 17A IL 17F IL 17A F TGF 6 IL 21 IL 22 ROR yt Foxp3 ST
51. Violet 405 nm Laser Uncompensated Compensated 4 2 Comp eFluor 450 A 10 102 103 104 10 eFluor 450 A Tow 102 Comp eFluor 605 A 102 103 eFluor 605 A TM eFluor 650 103 10025 Comp eFluor 650 A 104 105 102 10 eFluor 650 A 2 PE PE CD4 LSRII 405nm 488nm 633nm Blue 488 nm Laser Red 633 nm Laser Uncompensated Compensated Uncompensated Compensated 0 102 109 104 10 102 1024 10 Comp FITC A Comp APC A 102 10 104 10 0 102 Alexa Fluor 700 A 102 10 8 Comp Alexa Fluor 700 A 1007 80 40 i i i 60 i 1 1 i 20 i 0 10 102 10 102 104 109 10 102 102 104 10 0102 10 104 10 PerCP Cy5 5 A Comp PerCP Cy5 5 A APC Alexa Fluor 750 A Comp APC Alexa Fluor 750 A 0102 102 104 105 Comp PE Cy A FLOW CYTOMETRY Secti
52. Cat No 17 0081 The histograms top demonstrate staining of CD205 on CD11c CD8a cells pink histogram and CD11c CD8a cells blue histogram as gated in the contour plot below T CD83 CCR7 CD80 CD86 CD40 CD54 Plasmacytoid DC Plasmacytoid DC pDC Conventional DC pDC pDC ILT7 CD123 IL 3 pDC SiglecH PDCA 1 eBioscience has an extensive selection of reagents for studying all subsets of dendritic cells including Langerhans cells and monocyte plasmacytoid and interstitial
53. 4 5 7 ys XPermeabilization Buffer CHR Ly 21 8 300 400Xg 4 5 9 Optional 1XPermeabilization Buffer 100 1 normal mouse rat serum 2 2ul 10 30 4C 11 1ml 1xXPermeabilization Buffer 12 300 400Xg 4 5 13 1ml Flow Cytometry Staining Buffer 14 300 400Xg 4 5 229 15 500ul Flow Cytometry Staining Buffer c eBioscience Fixation amp Permeabilization Kit Cat No 88 8823 eBioscience Foxp3 Staining Buffer Set Cat No 00 5523 Transcription factors Nuclear proteins Cytokines Secreted proteins Cytoplasmic proteins Intracellular Staining Buffer Selection Guide Components are also sold individually t
54. CD11b positive cells CD19 positive cells were further analyzed based on expression of CD23 and CD24 to identify the T1 T2 and follicular subsets 190 IgM and CD93 expression on each subset is shown Immature Transitional lgD FITC Cells Follicular pl hoc E CD5 B1 B B BB B BLIMP 1 B B XLA B Btk OF B
55. CD8 T Cells Un 0 102 104 109 Deeore60s5 0 103 io4 10 CD4 eFluor 450 CD4 T Cells CD44 APC 0 103 104 10 CD62L eFluor 605N5 eBioscience provides antibodies conjugated to the most complete panel of high quality fluorochromes and all the necessary support reagents to help you get the most out of your flow cytometry BUFFERS FOR FLOW CYTOMETRY Flow Cytometry Staining Buffer eFluor NC Flow Cytometry Staining Buffer Foxp3 Staining Buffer Set IC Fixation Buffer Permeabilization Buffer eBioscience 1X RBC Lysis Buffer SUPPORT FOR FLOW CYTOMETRY 7 AAD Viability Staining Solution Propidium lodide Staining Solution Anti Mouse CD16 32 blocks Fc binding Human Fc y R Binding Inhibitor blocks Fc binding Brefeldin A Solution Monensin Solution FLOW CYTOMETRY EM CELL WHERE IT ALL BEGINS PM i 1961 Til McCullough 4 HSC shaded histogram 2 22 CFUS
56. EERE MAES negativ entre DMSO anti CD95 0 06 1ug ml negative control 3 BLK 5 CO2 37 58 2 mu a 37 59 CO02 4 Actinomycin D Aphidocolin Cycloheximide Dexamethasone 5 Fluorouracil Hydroxyurea and Staurosporine C 3 Annexin V Kit for detection of
57. FITC 128K 64K U Un Un WY 64K 128K 192K 256K 101 102 103 104 10 64K 128K 192K 256K 10 102 102 104 10 FSC CD45 FITC FSC CD45 FITC 256K 256K 256K 192K 192K 192K 128K 128K 128K Gus 64K Un Un WY WY 64K 128K 192K 256K 101 10 102 104 10 1 2 3 4 5 64K 128K 192K 256K 10 102 102 104 10 FSC CD45 FITC FSC CD45 FITC FLOW CYTOMETRY Section Il Choosing Fluorochromes MULTICOLOR FLOW CYTOMETRY
58. Fluor 700 Fluor 750 450 NOTES MEMO MEMO _ full spectrum cell analysis e eBioscience HHHHHHHHHHHHUHHHHLH n 105 0013 1 24 8ORIXHHHHH 7F ES TEL 03 6430 4020 03 6430 4021 NOVEL SCIENCE Co Ltd T113 0033 7 2 1 TEL 03 5842 2901 FAX 03 5842 2905 nc ADT RAR ZEE WAKENYAKU CO LTD fi 606 8171 17 525 0029 945 1 TEL 077 568 2911 FAX 077 568 2944 075 721 8111 0774 65 2521 077 568 2481 078 306 6060 072 749 5200 http www wakenyaku co jp
59. 00 base pair DNA Nur77 staining of apoptotic cells Mouse thymocytes were prepared as a single cell suspension and either left unstimulated top or stimulated with PMA and ionomycin bottom for 2 hours at 37 C Samples were stained intracellularly with PE mouse 1461 isotype control Cat No 12 4714 pink histogram or PE anti mouse Nur77 Cat No 12 5965 blue histogram Intracellular staining was performed using the Foxp3 Staining Buffer Set Cat No 00 5523 BrdUTP TdT 5 2 BrdU BrdU BrdU APO BRDU 88 6671 DNA DNA 2
60. 2 Ivanov McKenzie BS Zhou L Tadokoro CE Lepelley A Lafaille JJ Cua DJ Littman DR orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL 17 T helper cells Cell 2006 Sep 22 126 6 1121 33 THE HUMORAL IMMUNE RESPONSE B B B BCR BCR B BCR BCR BCR B IL 7 FIt3L TSLP
61. 2009 2010 FLOW CYTOMETRY Q full spectrum cell analysis eBioscience FLOW CYTOMETRY MULTICOLOR THE FULL SPECTRUM CONCEPT E 1 5 3 5 E 7 48 9
62. 35 640 723 APC eFluor 780 633 635 640 780 APC Alexa Fluor 750 633 635 640 775 4 nie 1 eBioscience
63. 7 1ml 12X75mm 8 brown cap natural cap ETA 70 9 1ml x 106cells ml 12X75mm 10 300 400Xg 4 5 11 1ml Wash Buffer blue cap 2 300 400Xg 4 5 12 50ul DNA 13 37 60 DNA 15 NOTE DNA 22 24 37
64. AT 3 STAT 5 TGF 6 IL 6 IL 21 IL 23 TGF B IL 23 IL 21 Th17 IL 23 IL 12 p40 IL 17A TGF IL 6 Th17 IL 23 IL 23 Th17 eBioscience offers a complete portfolio of reagents for the analysis of Th17 related proteins including master regulator transcription factors cytokines and cell surface markers KEY CD161 Staining of IL 17A and IL 17F in Th17 polarized mouse splenocytes CD196 CCR6 BALB c splenocytes cultured for 3 days with anti CD3 CD28 anti IL 2 recombinant TGF B and IL 6 followed by treatment with Brefeldin A alone IL 17A left or with PMA lonomycin and Brefeldin A right then stained with anti mouse IL 17A PE Cat No 12 7177 and anti mouse IL 17F Alexa Fluor 647 IL 17F Cat No 51 7471 Cells in the lymphocyte gate were used for analysis ROR y t ESSENTIAL CD3 CD4 IL 6 IL 12 1L 23 p40 IL 21 Staining of IL 17A and IL 21 in Th17 polarized CD4 human PBMC CD4 T cells were sorted fro
65. H TISSUE PERIPHERAL BLOOD IMMUNOFLUORESCENT STAINING CELL SURFACE ANTIGENS INTRACELLULAR STAINING DETECTING APOPTOTIC CELLS JRCE GUIDE TECHNICAL NOTES LIGHT SCATTER PROPERTIES CHOOSING FLUOROCHROMES SPECTRAL OVERLAP amp COMPENSATION FLOW CYTOMETRY Section Cell Preparation General Notes 1 2 3 EFKAR ua aeg cci ERU 1 71 p www tissuedissociation com I A Accutase Enzyme Cell Detachment Medium eBioscience Cat No 00 4555 eBioscience Flow Cytometry Staining Buffer eBioscience Cat No 00 4222 15 or 50 ml conical centrifuge tubes 1
66. Validated for most cytokine proteins NT not tested FLOW CYTOMETRY Section Il Immunofluorescent Staining C 1 1 CD95 Fas induced apoptosis of human cells 2 Pharmacologic induction of apoptosis with Camptothecin using EOS9 1 mAb B Jurkat clone E6 1 ATCC Cat No TIB 152 10 FCS RPMI 1640 Functional Grade purified anti human CD95 Fas APO 1 clone EOS9 1 eBioscience Cat 16 0958 109 FCS 109 FCS RPMI 1640 1 mM stock solution of Camptothecin prepared in DMSO 1 109 FCS RPMI 1640 0 5x106 mL 2 4 6uM 1mM Camptothecin JOR 640 0 5X106 mL
67. derived dendritic cells KEY PRODUCTS CD85g ILT7 CD205 DEC205 CD317 PDCA 1 MHC I K SIINFEKL siglec H ESSENTIAL PRODUCTS CD11c CD80 CD86 CD207 Langerin CD209 DC SIGN T References Shortman K Liu Y J 2002 Mouse and human dendritic cell subtypes Nat Rev Immunol 2 151 161 Colonna M Trinchieri G Liu Y J 2004 Plasmacytoid dendritic cells in immunity Nat Immunol 5 1219 1226 Randolph G Ochando J Partida Sanchez S 2008 Migration of dendritic cell subsets and their precursors Annu Rev Immunol 26 293 316 Merad Ginhoux F Collin 2008 Origin homeostasis function of Langerhans cells and other langerin expressing dendritic cells Nat Rev Immunol 8 935 947 RESO FLOW CYTOMETRY PROTOCOLS CELL PREPARATION TISSUE CULTURE CELLS FRES
68. e Cat No 24 5544 eBioscienceTM Fixation Permeabilization Diluent 3part eBioscienceTM Fixation Permeabilization Concentrate 1part Fixation PermeabilizationO L 1ml Fixation Permeabilization 10X Permeabilization Buffer 1X 2 3ml Permeabilization Buffer CI 10X Permeabilization Buffer 1X 3 4ml Permeabilization Buffer 1 Section Preparation 2 Protocol A 1 A2 3 4 5 30 6043 4 6 300 400Xg
69. early apoptotic events Annexins Annexin Vik PS PS inner leaflet PS leaflet PS Ca2 Annexin V propidium iodide 7 AAD viabllty dyes AnnexinV AnnexinV Tt Anexin V PS propidium iodide 7 AAD
70. m normal human peripheral blood cultured for 3 days with anti CD3 28 and then for 3 days with recombinant IL 1 6 and IL 23 followed by treatment with Brefeldin A PMA and lonomycin Cells were then stained with anti human IL 17A PE Cat No 12 7179 and either anti human CD4 FITC Cat No 11 0049 left or anti human IL 21 Alexa Fluor 647 Cat No 51 7219 right Cells in the lymphocyte gate were used for analysis References Th17 Th17 IL 17A IL 17F IL Dong TH17 cells in development an updated view of their 17A IL 21 IL 22 molecular identity and genetic programming Nat Rev Immunol ae May 8 5 337 48 Review Harrington LE Hatton RD Mangan PR Turner H Murphy TL Th17 anes Murphy Weaver Interleukin 17 producing CD4 effector T CD161 CCR6 Th1 7 cells develop via a lineage distinct from the T helper type 1 and 2 Th17 lineages Nat Immunol 2005 Nov 6 11 1123 3
71. o 12 4714 PE Va24Ja18T Cat No 12 5806 T Type I NKT Classical CD3 TCR CD94 NKp46 semi invariant Type II NKT CD3TTCR mouse 14 18 Variant human 24 18 yes yes yes no B T NKp46 RORgt IL 22 NK NKT NKT T NK CD1d eBioscience offers a complete portfolio of reagents for the analysis of both NKand NKT cells by flow cytometry immunoassay and bioassay KEY PRODUCTS CD158a f CD160 Eomes NKp46 Vo24Jo18 TCR 96 of Max 96 of Max 102 102 10 CD160 Alexa Fluor 647 0102 102 10 105 NKp46 FITC ESSENTIAL PRODUCTS CD49b CD94 NK1 1 NKG2D Perforin CD3 T References NK1 1 T
72. on Spectral Overlap amp Compensation MULTICOLOR FLOW CYTOMETRY 1 0 2 CD4 FITC PE FITC PE
73. ult human fibroblasts by defined factors Cell 131 861 872 Bonnet D Dick J E 1997 Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive Stem Cells and Cancer hematopoietic cell Nat Med 3 730 737 1997 AMES EB TEES AML CD34 CD38 TI LOW CYTOMETRY h1 DECIPHERING Th17 CELLS T CD4 T CD4 T Th1 Th2 T 2 T IL 17 IL 17A 2000 IL 17A
Download Pdf Manuals
Related Search