User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/17: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(8 intermediate revisions by the same user not shown)
Line 39: Line 39:
|-
|-
!A
!A
|1A1
|style="background:red; color:white" |1A1
|1A2
|style="background:red; color:white" |1A2
|1A3
|style="background:red; color:white" |1A3
|1A4
|style="background:red; color:white" |1A4
|1A5
|style="background:red; color:white" |1A5
|1A6
|style="background:red; color:white" |1A6
|2A1
|style="background:blue; color:white" |2A1
|2A2
|style="background:blue; color:white" |2A2
|2A3
|style="background:blue; color:white" |2A3
|2A4
|style="background:blue; color:white" |2A4
|2A5
|style="background:blue; color:white" |2A5
|2A6
|style="background:blue; color:white" |2A6
|-
|-
!B
!B
|1B1
|style="background:red; color:white" |1B1
|1B2
|style="background:red; color:white" |1B2
|1B3
|style="background:red; color:white" |1B3
|1B4
|style="background:red; color:white" |1B4
|1B5
|style="background:red; color:white" |1B5
|1B6
|style="background:red; color:white" |1B6
|2B1
|style="background:blue; color:white" |2B1
|2B2
|style="background:blue; color:white" |2B2
|2B3
|style="background:blue; color:white" |2B3
|2B4
|style="background:blue; color:white" |2B4
|2B5
|style="background:blue; color:white" |2B5
|2B6
|style="background:blue; color:white" |2B6
|-
|-
!C
!C
|1C1
|style="background:red; color:white" |1C1
|1C2
|style="background:red; color:white" |1C2
|1C3
|style="background:red; color:white" |1C3
|1C4
|style="background:red; color:white" |1C4
|1C5
|style="background:red; color:white" |1C5
|1C6
|style="background:red; color:white" |1C6
|2C1
|style="background:blue; color:white" |2C1
|2C2
|style="background:blue; color:white" |2C2
|2C3
|style="background:blue; color:white" |2C3
|2C4
|style="background:blue; color:white" |2C4
|2C5
|style="background:blue; color:white" |2C5
|2C6
|style="background:blue; color:white" |2C6
|-
|-
!D
!D
|1D1
|style="background:red; color:white" |1D1
|1D2
|style="background:red; color:white" |1D2
|1D3
|style="background:red; color:white" |1D3
|1D4
|style="background:red; color:white" |1D4
|1D5
|style="background:red; color:white" |1D5
|1D6
|style="background:red; color:white" |1D6
|2D1
|style="background:blue; color:white" |2D1
|2D2
|style="background:blue; color:white" |2D2
|2D3
|style="background:blue; color:white" |2D3
|2D4
|style="background:blue; color:white" |2D4
|2D5
|style="background:blue; color:white" |2D5
|2D6
|style="background:blue; color:white" |2D6
|-
|-
!E
!E
|3A1
|style="background:orange; color:white" |3A1
|3A2
|style="background:orange; color:white" |3A2
|3A3
|style="background:orange; color:white" |3A3
|3A4
|style="background:orange; color:white" |3A4
|3A5
|style="background:orange; color:white" |3A5
|3A6
|style="background:orange; color:white" |3A6
|
|
|
|
Line 109: Line 109:
|-
|-
!F
!F
|3B1
|style="background:orange; color:white" |3B1
|3B2
|style="background:orange; color:white" |3B2
|3B3
|style="background:orange; color:white" |3B3
|3B4
|style="background:orange; color:white" |3B4
|3B5
|style="background:orange; color:white" |3B5
|3B6
|style="background:orange; color:white" |3B6
|
|
|
|
Line 123: Line 123:
|-
|-
!G
!G
|3C1
|style="background:orange; color:white" |3C1
|3C2
|style="background:orange; color:white" |3C2
|3C3
|style="background:orange; color:white" |3C3
|3C4
|style="background:orange; color:white" |3C4
|3C5
|style="background:orange; color:white" |3C5
|3C6
|style="background:orange; color:white" |3C6
|
|
|
|
Line 137: Line 137:
|-
|-
!H
!H
|3D1
|style="background:orange; color:white" |3D1
|3D2
|style="background:orange; color:white" |3D2
|3D3
|style="background:orange; color:white" |3D3
|3D4
|style="background:orange; color:white" |3D4
|3D5
|style="background:orange; color:white" |3D5
|3D6
|style="background:orange; color:white" |3D6
|
|
|
|
Line 153: Line 153:


* Supernatant is stored @ -20 °C and used for ELISA later on
* Supernatant is stored @ -20 °C and used for ELISA later on
* Remaining cells are used for viability staining. (See [[http://openwetware.org/wiki/User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17#Alamar_Blue_staining Alamar Blue staining]])
* Remaining cells are used for viability staining. (See [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17#Alamar_Blue_staining| Alamar Blue staining]])


====Alamar Blue staining====
====Alamar Blue staining====
Line 165: Line 165:


==Results==
==Results==
*{{todo| 1 D9 (P21)}}
===Alamar Blue===
*{{todo| 1 D12 (P27)}}
{| border="1"
*{{todo| 2 D9 (P21)}}
|+'''D9 (passage 21)'''
*{{todo| 2 D12 (P27)}}
!#1
*{{todo| 3 D9 (P21)}}
!<u>1</u><BR>HT31P
*{{todo| 3 D12 (P27)}}
!<u>2</u><BR>HT31P
!<u>3</u><BR>HT31P
!<u>4</u><BR>HT31
!<u>5</u><BR>HT31
!<u>6</u><BR>HT31
|-
!A (CTR)
|style="background:red" |1824
|style="background:red" |1982
|style="background:red" |1953
|style="background:green" |5125
|style="background:green" |5172
|style="background:green" |5939
|-
!B (CSE)
|style="background:red" |956
|style="background:red" |964
|style="background:red" |785
|style="background:green" |4465
|style="background:green" |5006
|style="background:green" |5815
|-
!C (cse +8p)
|style="background:red" |1075
|style="background:red" |1104
|style="background:red" |971
|style="background:green" |5017
|style="background:green" |5241
|style="background:green" |5526
|-
!D (cse+6Bnz)
|style="background:red" |761
|style="background:red" |674
|style="background:red" |645
|style="background:green" |4408
|style="background:green" |5385
|style="background:green" |4943
|-
|}
<BR>
{| border="1"
|+'''D9 (passage 21)'''
!#2
!<u>1</u><BR>HT31P
!<u>2</u><BR>HT31P
!<u>3</u><BR>HT31P
!<u>4</u><BR>HT31
!<u>5</u><BR>HT31
!<u>6</u><BR>HT31
|-
!A (CTR)
|style="background:red" |2232
|style="background:red" |2560
|style="background:red" |2506
|style="background:green" |6431
|style="background:green" |6394
|style="background:green" |7264
|-
!B (CSE)
|style="background:red" |1113
|style="background:red" |1050
|style="background:red" |998
|style="background:green" |6077
|style="background:green" |6513
|style="background:green" |7171
|-
!C (cse +8p)
|style="background:red" |1224
|style="background:red" |1024
|style="background:red" |1234
|style="background:green" |5606
|style="background:green" |6203
|style="background:green" |7559
|-
!D (cse+6Bnz)  
|style="background:red" |730
|style="background:red" |826
|style="background:red" |858
|style="background:green" |5309
|style="background:green" |4856
|style="background:green" |2518
|-
|}
<BR>
{| border="1"
|+'''D9 (passage 21)'''
!#3
!<u>1</u><BR>HT31P
!<u>2</u><BR>HT31P
!<u>3</u><BR>HT31P
!<u>4</u><BR>HT31
!<u>5</u><BR>HT31
!<u>6</u><BR>HT31
|-
!A (CTR)
|style="background:red" |2359
|style="background:red" |2494
|style="background:red" |2434
|style="background:green" |5865
|style="background:green" |6713
|style="background:green" |6843
|-
!B (CSE)
|style="background:red" |998
|style="background:red" |929
|style="background:red" |957
|style="background:green" |5129
|style="background:green" |5632
|style="background:green" |6518
|-
!C (cse +8p)
|style="background:red" |946
|style="background:red" |1033
|style="background:red" |1026
|style="background:green" |5562
|style="background:green" |5622
|style="background:green" |6125
|-
!D (cse+6Bnz)  
|style="background:red" |940
|style="background:red" |1123
|style="background:red" |982
|style="background:green" |6428
|style="background:green" |5590
|style="background:green" |4116
|-
|}
<BR>
{| border="1"
|+'''D12 (passage 27)'''
!#1
!<u>1</u><BR>HT31P
!<u>2</u><BR>HT31P
!<u>3</u><BR>HT31P
!<u>4</u><BR>HT31
!<u>5</u><BR>HT31
!<u>6</u><BR>HT31
|-
!A (CTR)
|style="background:red" |2151
|style="background:red" |2049
|style="background:red" |2021
|style="background:green" |7501
|style="background:green" |7411
|style="background:green" |7507
|-
!B (CSE)
|style="background:red" |773
|style="background:red" |848
|style="background:red" |814
|style="background:green" |5447
|style="background:green" |6630
|style="background:green" |6700
|-
!C (cse +8p)
|style="background:red" |718
|style="background:red" |769
|style="background:red" |769
|style="background:green" |4324
|style="background:green" |4835
|style="background:green" |6349
|-
!D (cse+6Bnz)  
|style="background:red" |877
|style="background:red" |874
|style="background:red" |1002
|style="background:green" |5387
|style="background:green" |7649
|style="background:green" |6039
|-
|}
<BR>
{| border="1"
|+'''D12 (passage 27)'''
!#2
!<u>1</u><BR>HT31P
!<u>2</u><BR>HT31P
!<u>3</u><BR>HT31P
!<u>4</u><BR>HT31
!<u>5</u><BR>HT31
!<u>6</u><BR>HT31
|-
!A (CTR)
|style="background:red" |1389
|style="background:red" |2124
|style="background:red" |1980
|style="background:green" |7432
|style="background:green" |7069
|style="background:green" |7977
|-
!B (CSE)
|style="background:red" |925
|style="background:red" |1021
|style="background:red" |972
|style="background:green" |4957
|style="background:green" |5760
|style="background:green" |6746
|-
!C (cse +8p)
|style="background:red" |960
|style="background:red" |1030
|style="background:red" |1069
|style="background:green" |5381
|style="background:green" |4675
|style="background:green" |6522
|-
!D (cse+6Bnz)  
|style="background:red" |767
|style="background:red" |877
|style="background:red" |1138
|style="background:green" |5216
|style="background:green" |6143
|style="background:green" |6754
|-
|}
<BR>
{| border="1"
|+'''D12 (passage 27)'''
!#3
!<u>1</u><BR>HT31P
!<u>2</u><BR>HT31P
!<u>3</u><BR>HT31P
!<u>4</u><BR>HT31
!<u>5</u><BR>HT31
!<u>6</u><BR>HT31
|-
!A (CTR)
|style="background:red" |2071
|style="background:red" |2158
|style="background:red" |2331
|style="background:green" |7252
|style="background:green" |7248
|style="background:green" |7881
|-
!B (CSE)
|style="background:red" |1098
|style="background:red" |953
|style="background:red" |1087
|style="background:green" |5078
|style="background:green" |5458
|style="background:green" |5958
|-
!C (cse +8p)
|style="background:red" |926
|style="background:red" |1324
|style="background:red" |1106
|style="background:green" |4599
|style="background:green" |4899
|style="background:green" |5503
|-
!D (cse+6Bnz)  
|style="background:red" |908
|style="background:red" |738
|style="background:red" |957
|style="background:green" |4963
|style="background:green" |4311
|style="background:green" |6101
|-
|}
<BR>


==Conclusion==
==Conclusion==
*{{done}} Ow..
Samples should have been been into the wells at random to prevent the cells to concentrate in the first samples.
 
===Related entries===
====Run 1: Ht31/Ht31P D9/D12====
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/10|Splitting cells 10Feb2010]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/12|Counting cells 12Feb2010]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/15|Putting to S<sup>0</sup> 15Feb2010]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16|Stimulating cells 16Feb2010]]


==Related topics==
==Related topics==

Revision as of 14:17, 18 March 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

Viability staining <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • The supernatant of 3 24-wells plates per donor are put on 1 96-wells plate

Materials & Methods

Materials

Method

Preparations ELISA

  • Take 200 μL of supernatant of each well and put it in 96-wells plate
    • Divide like shown below, (#plate (24-wells);horizontal lane; vertical lane)
96-wells plate
01 02 03 04 05 06 07 08 09 10 11 12
A 1A1 1A2 1A3 1A4 1A5 1A6 2A1 2A2 2A3 2A4 2A5 2A6
B 1B1 1B2 1B3 1B4 1B5 1B6 2B1 2B2 2B3 2B4 2B5 2B6
C 1C1 1C2 1C3 1C4 1C5 1C6 2C1 2C2 2C3 2C4 2C5 2C6
D 1D1 1D2 1D3 1D4 1D5 1D6 2D1 2D2 2D3 2D4 2D5 2D6
E 3A1 3A2 3A3 3A4 3A5 3A6
F 3B1 3B2 3B3 3B4 3B5 3B6
G 3C1 3C2 3C3 3C4 3C5 3C6
H 3D1 3D2 3D3 3D4 3D5 3D6
  • Supernatant is stored @ -20 °C and used for ELISA later on
  • Remaining cells are used for viability staining. (See Alamar Blue staining)

Alamar Blue staining

  • Remove remaining medium from 24-wells plates
  • Wash twice with PBS
  • Add per well 475 μL HBSS + 25 μL Alamar blue
    • For ~75 wells that's 35.625 mL HBSS + 1875 μL Alamar blue
  • Incubate ~30 min. 37 °C
  • Measure in Wallace 1420 Victor 2TM, excitation 544 nm emission 390 nm
  • This shows the metabolic (mitochondria) activity of cells

Results

Alamar Blue

D9 (passage 21)
#1 1
HT31P 		2
HT31P 		3
HT31P 		4
HT31 		5
HT31 		6
HT31
A (CTR) 1824 1982 1953 5125 5172 5939
B (CSE) 956 964 785 4465 5006 5815
C (cse +8p) 1075 1104 971 5017 5241 5526
D (cse+6Bnz) 761 674 645 4408 5385 4943


D9 (passage 21)
#2 1
HT31P 		2
HT31P 		3
HT31P 		4
HT31 		5
HT31 		6
HT31
A (CTR) 2232 2560 2506 6431 6394 7264
B (CSE) 1113 1050 998 6077 6513 7171
C (cse +8p) 1224 1024 1234 5606 6203 7559
D (cse+6Bnz) 730 826 858 5309 4856 2518


D9 (passage 21)
#3 1
HT31P 		2
HT31P 		3
HT31P 		4
HT31 		5
HT31 		6
HT31
A (CTR) 2359 2494 2434 5865 6713 6843
B (CSE) 998 929 957 5129 5632 6518
C (cse +8p) 946 1033 1026 5562 5622 6125
D (cse+6Bnz) 940 1123 982 6428 5590 4116


D12 (passage 27)
#1 1
HT31P 		2
HT31P 		3
HT31P 		4
HT31 		5
HT31 		6
HT31
A (CTR) 2151 2049 2021 7501 7411 7507
B (CSE) 773 848 814 5447 6630 6700
C (cse +8p) 718 769 769 4324 4835 6349
D (cse+6Bnz) 877 874 1002 5387 7649 6039


D12 (passage 27)
#2 1
HT31P 		2
HT31P 		3
HT31P 		4
HT31 		5
HT31 		6
HT31
A (CTR) 1389 2124 1980 7432 7069 7977
B (CSE) 925 1021 972 4957 5760 6746
C (cse +8p) 960 1030 1069 5381 4675 6522
D (cse+6Bnz) 767 877 1138 5216 6143 6754


D12 (passage 27)
#3 1
HT31P 		2
HT31P 		3
HT31P 		4
HT31 		5
HT31 		6
HT31
A (CTR) 2071 2158 2331 7252 7248 7881
B (CSE) 1098 953 1087 5078 5458 5958
C (cse +8p) 926 1324 1106 4599 4899 5503
D (cse+6Bnz) 908 738 957 4963 4311 6101


Conclusion

Samples should have been been into the wells at random to prevent the cells to concentrate in the first samples.

Related entries

Run 1: Ht31/Ht31P D9/D12

Related topics

20.109(S08):Testing_cell_viability_(Day3)



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17&oldid=400533"