AKOKA Codex High-Parameter Tissue Analysis Instructions

AKOKA Codex High-Parameter Tissue Analysis

Prepare Conjugation Reagents and Antibodies

•  Purify antibodies if needed (see User Manual)
• Measure and calculate concentration of Antibodies using pre-set IgG settings of Nanodrop
•  Calculate the volume of solution corresponding to 50 μg of antibody.
• Retrieve the following reagents now:
• Reduction Solutions 1 & 2
• Filter Blocking Solution
• Retrieve the following reagents after 30-min incubation for step 4.1.5 :
  • Conjugation Solution
  • Barcodes
•  Retrieve the following reagents after 2-hour incubation for step 4.1.8 :
  • Purification Solution
  • Antibody Storage Solution

Antibody stocks: Purified Antibodies in PBS or in a similar buffer. Antibodies should be free of carrier proteins, such as BSA, and other chemicals such as gelatin or glycerol.

Akoya Materials

•  CODEX® Conjugation Kit
•  Reduction Solution 1
• Reduction Solution 2
• Filter Blocking Solution
• Conjugation Solution
•  Purification Solution
• Antibody Storage Solution
• CODEX® Barcodes

Materials NOT Included in Kit

Biologics:

• Purified antibody(s)
  Consumables:

• 50kDa MWCO filter

• 1.5 mL screw-top sterile tube(s)

•  Nuclease-Free Molecular Biology Grade Water

• PBS

• 0.2 mL PCR tubes (for quality check, Section 4.2)

• Bucket of ice for antibodies
  Instrumentation:

• Centrifuge for 1.5 mL tubes

• NanoDrop™ spectrophotometer

• Vortex (Optional)

Reduce Purified Antibody

•  Label a 50kDa MWCO filter for each antibody.
•  Add 500 μL of Filter Blocking Solution to the top of each 50kDa MWCO filter.
• Spin down at 12,000g for 2 mins.
• Remove all liquid; discard both the liquid left on the top of the column and the flow-through solution. Use a pipette if necessary to remove all liquid on top of the column.
•  Add 50 μg of the antibody in a volume 100 μL or greater.
• Spin down tubes at 12,000 g for 8 mins. Discard flow-through.

•  Create the Antibody Reduction Master Mix based on the number of CODEX® antibody conjugates.
  Antibody Reduction Master Mix

  Number of Antibodies| 1| 2| 3| 4| 5| 6| 7| 8

Reduction Solution 1 [µL]

|

6.6

|

13.2

|

19.8

|

26.4

|

33

|

39.6

|

46.2

|

52.8

Reduction Solution 2 [µL]

|

275

|

550

|

825

|

1100

|

1375

|

1650

|

1925

|

2200

Total [µL]| 281.6| 563.2| 844.8| 1126.4| 1378| 1689.6| 1971.2| 2252.8

• Add 260 μL of the Antibody Reduction Master Mix to the top of each filter unit. Vortex for 2-3 seconds.
• Incubate at RT for 30 mins. Exceeding 30 mins will result in irreparable damage to antibodies.
• Spin down tubes at 12,000 g for 8 mins. Discard flow-through.
• Add 450 μL of Conjugation Solution to the top of each column.
• Spin down tubes at 12,000 g for 8 mins. Discard flow-through.

Prepare CODEX® Barcodes and Conjugate Antibody

•  Resuspend each Barcode in 10 μl of Molecular Biology Grade Water by pipetting up and down.
•  Add 210 μL of Conjugation Solution to each suspended Barcode. Mix by gentle pipetting. Set aside.
• Add the CODEX® Barcode Solution to the top of each filter. Close the lid and vortex for 3 seconds.
• Incubate for 2 hours at RT

 Purify and Collect Tagged Antibodies

•  Set aside 2-5 μL of the purified solution for QC and troubleshooting.
•  Spin down tubes at 12,000 g for 8 mins. Discard flow-through.
• Add 450 μL of Purification Solution to the top of each column.
• Spin down tubes at 12,000 g for 8 mins. Discard the flow-through.
• Repeat steps c and d two more times for a total of three purifications.
• After the third centrifugation, discard the flow-through solution.
• The top of the column will contain the remaining purified solution. The filter will contain the conjugated antibody solution.
• For each antibody, label a new tube and lid that can hold filter units..
• Add 100 µL of Antibody Storage Solution to each filter unit column.
• After it is labeled, place the new empty tube upside-down on top of the filter unit column.
• Invert the filter for collection into the new collection tube.
• Spin solution down at 3,000g for 2 mins. The final volume in the tube should be around 120 µL.
• Transfer the solution to a sterile, screw-top tube for storage at 4°C for up to 1 year.
• Do not use these antibodies for tissue staining for at least 2 days; if used for staining sooner, you may experience high levels of background nuclear staining.

Validating Custom-Conjugated Antibodies

• Dilute conjugated antibodies and the unconjugated antibody (control) to a final volume of 13 μL in Nuclease free water.
• Add 5 μL of NuPAGE™ LDS Sample Buffer to each sample.
• Add 2 μL case NuPAGE™ Sample Reducing Agent to each sample.
•  Denature at 95°C in a dry bath for 10 mins.
• Prepare buffer by adding 40 mL of NuPAGE MOPS SDS Running Buffer and 760 mL of of NuPAGE MOPS SDS Running Buffer.
•  Prepare gel according to manufactures instructions.
•  Pour buffer into gel tank.
• Add 5 μL of a pre-stained protein standard 3.5-260 kDa to the gel.
•  Add 20 μL of antibody to one well each.
•  Run the gel at 200 V for 30-40 mins until completion.
• Place in a container filled with ddH2O.
• Microwave the gel until the first bubbles form.
• Stain the gel with Novex SimplyBlue™ SafeStain.
• Microwave the gel again until the first bubbles form.
• Place the gel in a shaker for 10 mins.
• Wash the gel with ddH2O and leave it on the shaker until bands are visible.

Antibody

• 5 μL of each conjugated antibody from section 4.1.9a.
• 1 µg (usually corresponding to 2 µL) of unconjugated antibody to be used as control.

Materials NOT Included in Kit

Reagents:

• NuPAGE™ LDS Sample Buffer (4X)

• NuPAGE™ Sample Reducing Agent (10X)

• NuPAGE™ 4-12% Bis-Tris Protein Gels

• Novex™ Sharp Pre-Stained Protein Standard – 3.5-260 kDa

• XCell SureLock™ Mini-Cell Electrophoresis System

• NuPAGE™ MOPS SDS Running Buffer (20X)

• Novex™ SimplyBlue™ SafeStain

• ddH2O

• Nuclease-free water
  Instrumentation:

• 95°C dry bath

• Microwave

• Shaker

From left to right: the first column shows the protein standard, columns from the second to the fifth show bands of successfully barcode-conjugated antibodies. The last column shows the heavy and light chain bands from a control, unconjugated antibody.

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