TITLE: SPARC - Millipore Metabolic Rat Multiplex Bead Assay for Flow Cytometry
AUTHORS: Doris Kemler

[DESCRIPTION]
This protocol describes methods of performing a Milliplex Assay in half of a 384-well plate, with each half-plate accommodating 12 rats (at 5 samples per rat) and duplicate controls (24 x 4 wells).

Assay: Millipore Metabolic Rat, 10 Analytes

Assay detail: 10 beads

[BEFORE_START]
​Because the Milliplex Assay kits are designed for 96 well plates, for every four kits used, order one extra set of assay buffer, detection antibody and Streptavidin-PE since these are limiting when we create equivalent of 4 assays from one 96 well kit.

Follow the Setting up the BEADS for the Millipore Metabolic Rat Milliplex Assay protocol to prepare the beads, within a month of the assay.

[GUIDELINES]
Using half of a 384 well-plate for the assay is an effective format that manages the maximum number of samples economically, and allows collection of sufficient samples to fill half of the plate. The second half will be performed within 1 month of filling the first half of the plate.

[STEPS]
SECTION: Preparation
1. Remove the kits from the cold room and allow everything to come to Room temperature (about 30 min before starting the assay).

SECTION: Preparation
2. Switch on the Mini-Orbital Shaker (pictured below is: Bellco Biotechnology 7744-08096 0-1150 RPM Mini-Orbital Mixer Stirrer Shaker, but newer alternatives such as the Bioshake shaker from www.Qinstruments.com are also recommended) to warm up the bearings, set it to 6. Shut it off any time after 15 min.

  

SECTION: Preparation
3. Create labels on the DYMO Label Writer 450 Turbo printer using specific labels (Labtags Company, Cat#ED1F-079) for C24 Standard and serum matrix.The label template is named Millipore Meta Rat Standards and matrix tubes. Change the expiration dates to one month from the current date. 

If the label writer is not available, use colored round stickers that can be used to create the labels.

  

SECTION: Preparation
4. Get all the reservoirs you will need for the day and place them upside down on the bench (this protects them from room dust). 
 

SECTION: Preparation
5. Organize the samples in a frozen PCR tube freezer rack (IsoFreeze, product #16361) the way you will load them on your plate.

  

SECTION: Preparation
6. Prepare the 384 filter plate.Tape the plate to an inverted lid to protect the filter bottom. (This is done to protect the bottom of the filter plate which is delicate.) Do this for the duration of the assay. Cover half the plate with a plate sealer if you are only using half the plate. (Cut plate sealer in half).

SECTION: Preparation
7. Remove the metal cap protectors from the Serum Matrix, Standard and Bead diluents, tubes provided in the Kits using forceps. 

SECTION: Preparation
8. Reconstitute the Serum Matrix by adding 1 mL (Millipore A10).

SECTION: Preparation
9. Reconstitute the Standard by adding 167 µL (Millipore A10).

SECTION: Preparation
10. Set timer for 10 min. This is the minimum time for reconstitution before you can make the standard curve.

SECTION: Preparation
11. Place the pre-labeled empty tubes in a PCR tube rack (21 x 8 tube holders; orange rack), organizing tubes with the highest number placed on the top left as such: 

in row A​:  24/22/20/18/16/14/12/10/8/6/4/2/0
in row F​:   23/21/19/17/15/13/11/9/7/5/3/1/0  

SECTION: Making Standards
12. Open the Assay buffer and pour about half of the bottle into the reservoir (pre-labeled with "assay buffer"). 

 

SECTION: Making Standards
13. Using a p125 pipettor, add 25 µL to all tubes EXCEPT for tube 24 and 23.
SECTION: Making Standards
14. Prepare the pipette: Preset->Stepper->pre-step, 25µl, 2x, 9, 7.
 

SECTION: Making Standards
15. Add 15 µL in tube 23 using a normal pipette.

SECTION: Making Standards
16. Spin all of the tubes in the platefuge (PlateFuge, Benchmark Inc) to make sure all of the buffer is at the bottom with no bubbles present, roughly 1 min (up to 2 minutes is fine).    
 

  



SECTION: Making Standards
17. Mix the standard vial well from all sides.
SECTION: Making Standards
18. Centrifuge the standard vial in the platefuge.
SECTION: Making Standards
19. Use a P200 pipette to transfer the standard to the C24 tube. 
 

SECTION: Making Standards
21. Add 30 µL of tube C24 (standard) into tube C23 and pipette-mix four times after dispensing. 

SECTION: Making Standards
20. With a pipette, mix the C24 tube contents thoroughly, immediately prior to the next step.
SECTION: Making Standards
22. Make sure that tubes C24-C0 are in row A and C23-C0 are in row F (in the orange PCR rack). 
SECTION: Making Standards
23. Get out the special pipette box for standards dilution with the right spacing marked by the black lines and put 12 tips in each of the 2 rows.
SECTION: Making Standards
24. Using a 125 Eclip-tip pipette (E1-Clip, Thermo Scientific), go to programs and load “Dilute Stds” (yellow pipette)

  

SECTION: Making Standards
25. Perform the following steps with the Eclip-tip pipette:
SECTION: Making Standards
25.1. Pick up 2 tips (from the special pipette box).
 

SECTION: Making Standards
25.2. For second tube (C22/C21):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to second tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close second tube 
Open third tube
Pick up 2 tips (from the special pipette box)
SECTION: Making Standards
25.3. For third tube (C20/C19):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to third tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close third tube 
Open fourth tube
Pick up 2 tips (from the special pipette box)
SECTION: Making Standards
25.4. For fourth tube (C18/C17):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to fourth tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close fourth tube 
Open fifth tube
Pick up 2 tips (from the special pipette box)
SECTION: Making Standards
25.5. For fifth tube (C16/C15):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to fifth tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close fifth tube 
Open sixth tube
Pick up 2 tips (from the special pipette box)
SECTION: Making Standards
25.6. For sixth tube (C14/C13):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to sixth tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close sixth tube 
Open seventh tube
Pick up 2 tips (from the special pipette box)
SECTION: Making Standards
26. Remove C4 from the even number row and C0 from the odd number row (these tubes will not be used). We do not use the C4 dilution.
SECTION: Making Standards
27. Flip the order of the even tubes so that the numbers are organized as such: 
C0/C2/C6/C8/C10/C12/C14/C16/C18/C20/C22/C24
C23/C21/C19/C17/C15/C13/C11/C9/C7/C5/C3/C1
SECTION: Making Standards
25.7. For seventh tube (C12/C11):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to seventh tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close seventh tube 
Open eighth tube
Pick up 2 tips (from the special pipette box) 
SECTION: Making Standards
25.10. For tenth tube (C6/C5):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to tenth tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close tenth tube 
Open eleventh tube
Pick up 2 tips (from the special pipette box) 
SECTION: Making Standards
25.9. For ninth tube (C8/C7):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to ninth tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close ninth tube 
Open tenth tube
Pick up 2 tips (from the special pipette box) 
SECTION: Making Standards
25.8. For eighth tube (C10/C9):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to eighth tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close eighth tube 
Open ninth tube
Pick up 2 tips (from the special pipette box) 
SECTION: Making Standards
25.11. For eleventh tube (C4/C3):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to eleventh tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close eleventh tube 
Open twelfth tube
Pick up 2 tips (from the special pipette box) 
SECTION: Making Standards
25.12. For twelfth tube (C2/C1):
Put the tips to the bottom of the first tube
Trigger, mix (1/2)
Trigger, mix (2/2)
Beep
Touch off on sides
Move to twelfth tube bottom
Trigger, mix (1/2)
Trigger, mix (2/2)
Pull up bit above liquid with tip touching side
Trigger to dispense (25µl)
Tip over bucket
Trigger
Trigger, eject
Close twelfth tube 
 


SECTION: Loading the Half-filled 384-well Plate
28. Prepare samples.
 

SECTION: Loading the Half-filled 384-well Plate
29. Prepare standard tubes - open lids and put rack cover over the rack. 

SECTION: Loading the Half-filled 384-well Plate
30. Prepare beads - vortex mixed beads (check pellet) and sonicate for 10 s (up to 15 seconds). 

 

SECTION: Loading the Half-filled 384-well Plate
31. Prepare standards.
SECTION: Loading the Half-filled 384-well Plate
31.1. Remove/cut C4 dilution tube from the even dilutions and the C0 from the odd dilutions (neither are used). 
SECTION: Loading the Half-filled 384-well Plate
32. Fill the trays with the Matrix and Assay Buffer. 
SECTION: Loading the Half-filled 384-well Plate
31.2. Organize the even dilution row so that they list 0-24 when reading from left to right. Do not flip the odd dilutions. 

Plate numbers should read like: 
0 2 6 8 10 12 14 16 18 20 22 24
23 21 19 17 15 13 11 9 7 5 3 1
SECTION: Loading the Half-filled 384-well Plate
33. Prepare plates. 
SECTION: Loading the Half-filled 384-well Plate
33.1. Turn on vacuum (Model #WP6111560 Vacuum Pump, Billerica MA, 01821) and ensure it reads about -20Hg.

  

SECTION: Loading the Half-filled 384-well Plate
33.2. Before placing filter plate onto the vacuum manifold, remove metal support and wet the blue silicone rim of the manifold with water. 
SECTION: Loading the Half-filled 384-well Plate
33.3. Remove the bottom cover (plate lid) protecting the plate. 
SECTION: Loading the Half-filled 384-well Plate
33.4. Vacuum liquid out of the plate. 
SECTION: Loading the Half-filled 384-well Plate
33.5. Tap plate on stack of paper towels, until towels no longer show wet spots. 
SECTION: Loading the Half-filled 384-well Plate
33.6. Tape the protecting plate back on. 
SECTION: Loading the Half-filled 384-well Plate
34. Load Matrix.
SECTION: Loading the Half-filled 384-well Plate
34.1. Load 12 tips on the 125uL Ecliptip pipette. 
SECTION: Loading the Half-filled 384-well Plate
34.2. Fill the matrix tray with matrix solution. 
SECTION: Loading the Half-filled 384-well Plate
34.3. Select program "half 384-ZT matrix".  (There is a pre Step.)
SECTION: Loading the Half-filled 384-well Plate
34.4. Load matrix in rows AB and OP (shaded green in image below). Cover row A and B immediately after loading matrix.
 
  

SECTION: Loading the Half-filled 384-well Plate
35. Load Assay. 
SECTION: Loading the Half-filled 384-well Plate
35.1. Fill assay tray with assay buffer. 
SECTION: Loading the Half-filled 384-well Plate
35.2. Select program "half 384-ZT assay". (There is a pre Step.)
SECTION: Loading the Half-filled 384-well Plate
35.3. Load assay buffer in rows C to N (shaded in blue below). 

  

SECTION: Loading the Half-filled 384-well Plate
36. Load standards. (No pre step.)
SECTION: Loading the Half-filled 384-well Plate
36.1. Select program "half 384-ZT Std".
SECTION: Loading the Half-filled 384-well Plate
36.2. Load even row (0-24) in row A and O. 
Load odd row (23-1) in row B and P. 

  

SECTION: Loading the Half-filled 384-well Plate
37. Load samples. 
SECTION: Loading the Half-filled 384-well Plate
37.1. Select program "half 384 samples" and be sure to use only 5 tips with the pipette at the time.
SECTION: Loading the Half-filled 384-well Plate
37.2. Load samples in row C - load 7.5ul of the first half of sample in columns 1-5 and 7.5ul of the second half of sample in columns 6-10. 

  

SECTION: Loading the Half-filled 384-well Plate
37.3. Continue loading samples in rows D-N following the column structure from the previous step (first-half samples in columns 1-5 and second-half samples in columns 6-10). 

  

SECTION: Loading the Half-filled 384-well Plate
38. Load beads. 
 

SECTION: Loading the Half-filled 384-well Plate
38.1. Select program "half 384ZT beads". (There is a pre Step.)
SECTION: Loading the Half-filled 384-well Plate
38.2. Load beads in all rows A-P. 
 

SECTION: Loading the Half-filled 384-well Plate
38.3. Put lid back and tape it back on plate. 
SECTION: Prepare Bioshake
40. Tape entire plate with protection lid on the shaker. The Bioshake is one of the best shakers available and our experience is that regular lab bench shakers are not adequate for proper dispersal of the small number of beads in each well of a 384 well plate. THIS IS A CRITICAL STEP.
 

 

SECTION: Prepare Bioshake
41. Put aluminum foil over the plate TO PROTECT FROM LIGHT.
SECTION: Prepare Bioshake
42. Put the shaker in the cold room. 
SECTION: Prepare Bioshake
43. Lock the card wheels IF THE SHAKER IS ON A CART.
SECTION: Prepare Bioshake
44. Plug shaker in and turn computer on (some Bioschake devices can be run indepenendent of a computer but some (ie the ones generally used on automation systems) require a computer. 
SECTION: Prepare Bioshake
45. Start shaker at 2400rpm.
SECTION: Prepare Bioshake
46. Incubate for 960 min (up to 18 hours). Turn off monitor of computer. 

SECTION: Cleaning/Retrieving reagents
47. Return mixed beads to kit (previously in reservoir tray) and refrigerate. 
 

SECTION: Cleaning/Retrieving reagents
48. Put leftover serum matrix in a tube and label/date. 
SECTION: Cleaning/Retrieving reagents
49. Label/date and store leftover C23 and C24 standard solutions at -20 °C.

SECTION: Cleaning/Retrieving reagents
51. Check wash buffer. 
 

SECTION: Cleaning/Retrieving reagents
50. Pour extra assay buffer back into original container. 
SECTION: Cleaning/Retrieving reagents
52. Wash reservoirs and leave them to dry. 
SECTION: Cleaning/Retrieving reagents
53. Make notes regarding the plate. 
SECTION: Preparing for Detection
54. [Second Day, after 16-18 hours]

Warm kit (and last kit just in case) and wash buffer to Room temperature. 

SECTION: Preparing for Detection
55. Pour 400-500mL of wash buffer into short plastic bottle for the multidrop. 
SECTION: Preparing for Detection
56. Locate the shaker in the cold room. 
SECTION: Preparing for Detection
56.1. Turn computer monitor back on and stop shaker. (only if shaker is computer operated modle, otherwise just suspend operation) 
SECTION: Preparing for Detection
56.2. Shut down the computer. 
SECTION: Preparing for Detection
57. Bring the shaker back to the lab and set it up. 
SECTION: Preparing for Detection
57.1. Turn on the computer and start Bioshake software. 
SECTION: Preparing for Detection
57.2. Put the brakes on. 
SECTION: Preparing for Detection
58. Prepare the multidrop. Check both power buttons are switched on, that the primer container is in the correct position and the cover is closed right over the tubing.  

Ensure the following is correct: 
NO NOZZLES ARE CLOGGED
SET SELECTOR FOR 384
SET COLUMNS FOR 12
SET VOLUME FOR 75UL
SECTION: Preparing for Detection
59. Get reservoir out for detection AB and detection AB bottle. 
SECTION: Preparing for Detection
60. Set program on Ecliptip pipette to Preset>stepper>pre-step, 15.6µl, 8x, 9, 7.
SECTION: Preparing for Detection
61. Turn vacuum on and perform the following (using it at about -20Hg): 
SECTION: Preparing for Detection
61.1. Before placing filter plate onto the vacuum manifold, remove metal support and wet the blue silicone rim of the manifold with water. 

  

SECTION: Preparing for Detection
61.2. Vacuum liquid out of the plate. 
SECTION: Preparing for Detection
61.3. Blot button of the plate on paper towels several times, until towel is no longer wet. 
SECTION: Preparing for Detection
61.4. Put the plate on the multidrop. 
SECTION: Preparing for Detection
61.5. Add wash buffer using bottle, push start button. 
SECTION: Preparing for Detection
61.6. Vacuum liquid out, blot on paper until no longer wet. [you will repeat the wash 4x below]
SECTION: Preparing for Detection
61.7. Repeat wash: add wash buffer (1/4)
SECTION: Preparing for Detection
61.8. Repeat wash: vacuum liquid out, blot on paper until no longer wet (1/4)
SECTION: Preparing for Detection
61.9. Repeat wash: add wash buffer (2/4)
SECTION: Preparing for Detection
61.10. Repeat wash: vacuum liquid out, blot on paper until no longer wet (2/4)
SECTION: Preparing for Detection
61.11. Repeat wash: add wash buffer (3/4)
SECTION: Preparing for Detection
61.12. Repeat wash: vacuum liquid out, blot on paper until no longer wet (3/4)
SECTION: Preparing for Detection
61.13. Repeat wash: add wash buffer (4/4)


SECTION: Preparing for Detection
61.15. Repeat wash: vacuum liquid out, blot on paper until no longer wet (4/4)
SECTION: Preparing for Detection
62. Use yellow Ecliptip pipette to add 15.6 µL to all wells. 
 


SECTION: Preparing for Detection
61.14. Repeat wash: before last vacuum, pour detection AB in the large reservoir. (4/4)
SECTION: Preparing for Detection
63. Put lid back on and tape it on well using tape. 
SECTION: Preparing for Detection
64. Cover plate with aluminum foil and shake for 30 min at 2400rpm. 

SECTION: Preparing for Detection
65. Pour 3.5 mL in reservoir.

SECTION: Preparing for Detection
67. Use yellow Ecliptip pipette to add 15.6 µL  to all wells. 
 


SECTION: Preparing for Detection
66. Stop shaker.
SECTION: Preparing for Detection
68. Cover plate with aluminum foil and shake for 30 min at 2400rpm. 

SECTION: Preparing for Detection
69. Turn on/set up the Attune Cytometer

  

SECTION: Preparing for Detection
70. After shaking, turn off the skaker, remove plate and change speed to 1600rpm.
SECTION: Preparing for Detection
69.1. Start up and run performance test.
SECTION: Preparing for Detection
69.2. Load up template named "Mili-metabolic-rat-plate-384_well_half".
SECTION: Preparing for Detection
71. Vacuum off the contents of the plate.
SECTION: Preparing for Detection
72. Add 75 µL. 
SECTION: Preparing for Detection
73. Wash three more times:
SECTION: Preparing for Detection
73.1. Vacuum off the contents of the plate. (1/3)
SECTION: Preparing for Detection
73.2. Add 75 µL. (1/3)
SECTION: Preparing for Detection
73.3. Vacuum off the contents of the plate. (2/3)
SECTION: Preparing for Detection
73.4. Add 75 µL. (2/3)
SECTION: Preparing for Detection
73.5. Vacuum off the contents of the plate. (3/3)
SECTION: Preparing for Detection
73.6. Add 75 µL. (3/3)
SECTION: Preparing for Detection
75. Blot on paper towel. 
SECTION: Preparing for Detection
76. Wash twice more: 
SECTION: Preparing for Detection
76.1. Add 75 µL. (1/2)
SECTION: Preparing for Detection
76.2. Vacuum off the contents of the plate. (1/2)
SECTION: Preparing for Detection
76.3. Add 75 µL. (2/2)
SECTION: Preparing for Detection
76.4. Vacuum off the contents of the plate. (2/2)
SECTION: Preparing for Detection
77. Blot plate very well, until no wet spots show up on paper towels.
SECTION: Preparing for Detection
78. Add 75 µL. 
SECTION: Preparing for Detection
79. Shake plate at 1600rpm for 10 min. 

SECTION: Preparing for Detection
80. While the plate is shaking, get a box full of tips and a 384 v-bottom plate. Label the plate with the plate number and cover the second half of the plate with plate sealer. (Note: the second half of the plate is not used)
SECTION: Preparing for Detection
81. Set the yellow Ecliptip pipette program to "Transfer to 384" (this program will mix and pull 75 µL of probe). 

SECTION: Preparing for Detection
82. Change volume on the multi drop to 25 µL. 

SECTION: Preparing for Detection
83. Fill filter plate with 25 µL. 
SECTION: Preparing for Detection
84. Reattach filter plate to the shaker.
SECTION: Preparing for Detection
85. Shake for 3 min. Minutes

SECTION: Preparing for Detection
86. Start the time shake 5 seconds. 
SECTION: Preparing for Detection
87. Transfer the 25 µL probe to the 384 v-bottom plate. 

SECTION: Preparing for Detection
88. For repeat, hit 8 times tab and click on "5 sec". Repeat until plate is done. 
SECTION: Preparing for Detection
89. Put plate in Attune plate holder.  (Note the Attune plate holder cannot use filterplates becuase of the calibration bar that sits below the plate. This is the reason the filter plate has to be transferred to the regular plate. If you use the Beckman CYtoflex however, you do not need to transfer the filter plate as it can be placed directrly onto the Cytoflex cytometer.
SECTION: Cleaning Up
90. Put filter plate back to the multi drop.
SECTION: Cleaning Up
91. Change volume to 75µl.
SECTION: Cleaning Up
92. Add 75 µL to the plate.

SECTION: Cleaning Up
93. Put plate back to inverted plate.
SECTION: Cleaning Up
94. Put cover back on and tape it to the plate.
SECTION: Cleaning Up
95. Seal the plate with parafilm.
SECTION: Cleaning Up
96. Wrap the plate in aluminum foil.
SECTION: Cleaning Up
97. Label plate with plate number and date.
SECTION: Cleaning Up
98. Put the plate in fridge.
SECTION: Cleaning Up
99. Put the Millipore metabolic rat kit back in the cold room.
SECTION: Cleaning Up
100. Wash the reservoirs.
SECTION: Cleaning Up
101. Clean the multi drop machine with H2O. No water drops should be in the tubes anymore.
SECTION: Cleaning Up
101.1. Push the black guard backwards.
SECTION: Cleaning Up
101.2. Take the tubes out of the bracket.
SECTION: Prepare Bioshake
39. Bioshake: Q-Instruments, Germany.
Qcom, 2400 rpm for 25 µl
            1600 rpm for 75 µl

    

SECTION: Preparing for Detection
74. Vacuum off the contents of the plate. (3/3)