May 08, 2020

Public workspaceMaxpar Antibody Labeling for Imaging Mass Cytometry

DOI
dx.doi.org/10.17504/protocols.io.bf5jjq4n
  • 1University of Florida;
  • 2University of Zürich
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Marda Jorgensen
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DOI: dx.doi.org/10.17504/protocols.io.bf5jjq4n
Protocol Citation: Marda Jorgensen, Michelle Daniel 2020. Maxpar Antibody Labeling for Imaging Mass Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.bf5jjq4n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 07, 2020
Last Modified: October 02, 2020
Protocol Integer ID: 36747
Keywords: HuBMAP, HuBMAP, TMC-Florida/Zurich, Imaging Mass Cytometry
Abstract
Version 11 of the antibody labelling protocol published by Fluidigm.
Guidelines
Buffers and lanthanides: 4 °C
Polymers: –20 °C with provided desiccant in a sealed container
Materials
MATERIALS
ReagentPurified IgG or polyclonal: glycerol-free and carrier-free (no BSA hydrolyzed protein or gelatin
ReagentCentrifugal Filter Unit: 3 kDa Amicon® Ultra 500 μL V bottomMerck Millipore (EMD Millipore)Catalog #UFC500396
ReagentCentrifugal Filter Unit: 50 kDa Amicon Ultra 500 μL V bottomMerck Millipore (EMD Millipore)Catalog #UFC505096
ReagentMicrocentrifuge
Reagent37 °C water bath or heat block (for 2 mL Eppendorf® tubes)
Reagent0.5 M TCEP: Pierce™ Bond-Breaker® TCEP SolutionThermo ScientificCatalog #77720
ReagentAerosol barrier (filter) pipette tips (200uL)
ReagentAntibody Stabilizer (supplemented with 0.05% sodium azide after purchase)Candor Bioscience GmbHCatalog #131050
Safety warnings
Before handling chemicals, refer to the safety data sheet (SDS) provided by the manufacturer and observe all relevant precautions.
Before start
Antibody clones selected for conjugation are first validated for adequate sensittivity and expected staining patterns using immunofluorescence microscopy.

Maxpar Polymer is moisture-sensitive. Equilibrate polymer (stored at –20 °C) to room temperature before opening to avoid moisture condensation.
• Use filter tips in all pipetting steps to prevent cross-contamination between metal stocks and reagents.
• Before starting the Maxpar protocol, verify the purified, carrier-free antibody concentration by NanoDrop™ after blanking against the buffer they are suspended in. The composition of the buffer can be found on the technical data sheet supplied by the antibody vendor.
• Loading of the polymer and partial reduction of the antibody should be performed simultaneously (see Figure 1). It is imperative, however, not to exceed the recommended reduction time, and not to allow the partially reduced antibody to remain free of the loaded polymer.
Preload the polymer with lanthanide.
Preload the polymer with lanthanide.
1 Spin the polymer tube for 10 seconds in a microfuge to ensure that the reagent is at the bottom of the tube.
2 Resuspend the polymer with 95 μL of L-Buffer.
3 Mix thoroughly by pipetting.
4 Add 5 μL of lanthanide metal solution to the tube (final concentration: 2.5 mM in 100 μL).
5 Mix thoroughly by pipetting.
6 Incubate at 37 °C for 30−40 minutes in a water bath or heat block. During incubation, proceed immediately to Step 7 and begin buffer exchange and partial reduction of antibody.


Perform buffer exchange and partially reduce the antibody.
Perform buffer exchange and partially reduce the antibody.
7 Add 100 μg of stock antibody in up to 400 μL R-Buffer to a 50 kDa filter.
8 Centrifuge at 12,000 x g for 10 minutes at room temperature (RT).
9 During centrifugation, dilute 0.5 M TCEP stock to 4 mM in R-Buffer by mixing 8 μL of 0.5 M TCEP stock with 992 μL of R-Buffer. For each antibody being labeled, 100 μL of 4 mM TCEP-R-Buffer is required.
10 Discard column flow-through from centrifugation.
11 Add 100 μL of the 4 mM TCEP-R-Buffer to each antibody and mix by pipetting.
12 Incubate at 37 °C in a water bath or heat block for 30 minutes. Proceed to Step 13 during the 30-minute incubation.
Note
If the stock antibody concentration is too dilute to add the desired amount of antibody, pre-concentrate it in the same filter before adding the R-Buffer.

Note
IMPORTANT Do not exceed 30 minutes! Proceed immediately to Step 17 after 30 minutes and begin purifying the partially reduced antibody.

Purigy the lanthanide-loaded polymer.
Purigy the lanthanide-loaded polymer.
13 Add 200 μL of L-Buffer to a 3 kDa filter.
14 Add the 100 μL metal-loaded polymer mixture to the filter containing the 200 μL L-Buffer to the wash.
15 Centrifuge at 12,000 x g for 25 minutes at RT.
16 Repeat the wash by adding 400 μL of C-Buffer to the filter and centrifuge at 12,000 x g for 30 minutes at RT.
Note
Purify the lanthanide-loaded polymer at the same time that the antibody is being reduced (Step 12).

Purify the partially reduced antibody.
Purify the partially reduced antibody.
17 Retrieve the 50 kDa filter containing the partially reduced antibody from the 37 °C water bath or heat block.
18 Add 300 μL of C-Buffer to the 50 kDa filter to wash the antibody.
19 Centrifuge at 12,000 x g for 10 minutes at RT. (A second microcentrifuge could be used at this step to avoid timing conflict with the polymer wash.)
20 Discard flow-through.
21 Repeat the wash by adding 400 μL of C-Buffer to the filter and centrifuge at 12,000 x g for 10 minutes at RT.
Retrieve the purified partially reduced antibody and lanthanide-loaded polymer.
Retrieve the purified partially reduced antibody and lanthanide-loaded polymer.
22 Retrieve 3 kDa filter containing the purified lanthanide-loaded polymer from the centrifuge and discard column flow-through.
23 Retrieve 50 kDa filter containing the purified partially reduced antibody from the centrifuge and discard column flow-through.

Conjugate the antibody with lanthanide-loaded polymer.
Conjugate the antibody with lanthanide-loaded polymer.
24 Using a pipette, resuspend the lanthanide-loaded polymer in 60 μL of C-Buffer (total volume ~80 μL).
25 Transfer the resuspended contents to the corresponding partially reduced antibody in the 50 kDa filter (final conjugation volume ~100 μL).
26 Mix gently by pipetting.
27 Incubate at 37 °C for 90 minutes.

Wash the metal-conjugated antibody.
Wash the metal-conjugated antibody.
28 Add 200 uL of W-buffer to the 100 uL antibody conjugation mixture.
29 Centrifuge at 12,000 x g for 10 minutes.
30 Discard flow-through.
31 Repeat wash three more times with W-Buffer to a toal volume of 400 uL (for a total of four washes with W-Buffer)
Determine yield.
Determine yield.
32 After the final wash with W-buffer, add ~80 μL of W-buffer to the 50 kDa filter to dilute the conjugate (in ~20 μL) to a volume of 100 μL. Pipette to mix and rinse the walls of the filter.
33 Quantify the conjugated antibody by measuring the absorbance at 280 nm against a W-Buffer blank (expected recovery is 60%).
34 Calculate the volume of antibody stabilization buffer (supplemented with 0.05% sodium azide after purchase) required to obtain a final concentration of 0.5 mg/mL.
35 Centrifuge the 50 kDa filter at 12,000 x g for 10 minutes to remove the W-Buffer.
Recover and store the metal-conjugated antibody.
Recover and store the metal-conjugated antibody.
36 Add the calculated volume of antibody stabilization buffer (supplemented with 0.05% sodium azide after purchase) minus the residual volume (~20 μL) to the 50 kDa filter to obtain a final concentration of 0.5 mg/mL of conjugated antibody.
37 Invert the 50 kDa filter over to a new collection tube (see Figure 2).
38 Centrifuge the inverted filter/collection tube assembly at 1,000 x g for 2 minutes.
39 Store at 4 °C until ready to titrate.
40 Titrate the antibody. We recommend titrating the antibody with relevant positive and negative controls for the experimental system in which the antibody will be used. Set up the titration as follows: 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.62 μg/mL, 0.31 μg/mL, 0.16 μg/mL, 0 μg/mL.
41 After the conjugated antibody has been titrated on the CyTOF® instrument, if necessary dilute it to the optimum working concentration in stabilization buffer and store it at 4 °C.