Enrichment and Detection of Antigen-Binding B Cells for Mass Cytometry
Abstract
:1. Introduction
2. Materials and Methods
- Isolation of PBMC’s from Whole Blood
- Collect 30–50 mL of heparinized blood collection tubes
- Dilute heparinized blood 1:1 with sterile, room temperature magnesium and calcium-free phosphate-buffered saline (PBS).
- In a 50 mL conical tube, add 10–15 mL of room temperature Ficoll-Paque PLUS.
- Carefully layer 30–35 mL of the diluted blood on the top of the Ficoll-Paque at the lowest speed setting, being careful not to mix the two.
- Centrifuge at 400× g for 30 min at room temperature with the brake turned off.
- Using a serological pipette remove the mononuclear layer (buffy coat) and transfer to a clean 50 mL conical. Add at least 3 times the volume of PBS to the cell suspension.
- Centrifuge cells at 400× g for 10 min at room temperature with the brake on.
- Remove supernatant and resuspend cells in 10 mL PBS. Count cells and determine viability using a hemocytometer or automated cell counter.
- Adjust cell concentration to 3 × 107 cells/1 mL CSM buffer and place on ice until ready to begin processing.
- Viability Dye
- Centrifuge 30 × 106 cells at 400× g for 5 min at room temperature and aspirate supernatant.
- Wash cells 1× with 1 mL sterile PBS
- Do not use any solutions with serum or protein content until after cisplatin incubation.
- Spin down at 400× g for 5 min at room temperature and aspirate supernatant.
- Resuspend cells in serum-free RPMI at 3 × 106 cells per 1 mL RPMI.
- Add 1 uL (25 mM stock) of Cisplatin per 1 mL of serum-free RPMI and vortex samples briefly to mix thoroughly (25 uM final concentration).
- Incubate for 1 min at RT before quenching solution with 2 mL CSM or complete RPMI per 1 mL of cisplatin-cell solution.
- It is necessary to quench with a solution that contains protein to bind extraneous cisplatin
- Spin cells at 400× g for 5 min at room temperature and aspirate supernatant.
- Enrichment of antigen-reactive B cells
- Wash cells once with 1 mL CSM. Centrifuge cells at 400× g for 5 min and aspirate supernatant.
- Resuspend cells in 25 uL Fc Block and allow them to bind for 5 min on ice. Do not wash off.
- Add 1 mL CSM to cells before adding antigen(s) at their pre-determined amount. Vortex briefly to mix and incubate on ice for 15 min in the dark.
- Note: Biotinylated and FITC conjugated antigens should be titrated to determine the optimal concentrations needed to ensure detection and separation of binding cells from non-specific bystander cells. Typically, a good starting concentration is between 0.025 ug and 1 ug per 1 × 106 cells. (See Smith et al. for detailed protocol [10])
- Eagle-Insulin-biotin (0.4 mg/mL stock, commercially available): Use 0.035 ug/1 × 106 cells = 1.05 ug/ 30 × 106 cells = 2.63 uL
- Tetanus-Toxoid-FITC (2 mg/mL stock, FITC labeled according to manufacturer’s instructions): Use 1 ug/1 × 106 cells = 30 ug /30 × 106 cells. = 15 uL
- Wash cells 2× with 1 mL cold CSM at 400× g for 5 min before aspirating supernatant.
- During centrifugation prepare 1.6% PFA solution using the following steps:
- Break open an unopened ampule of 16% paraformaldehyde and sterile filter using a 0.45 μm syringe filter into a 15 mL conical tube.
- Dilute to 1.6% PFA by adding 9 mL of CSM to the 1 mL of 16% PFA. Wrap conical tightly in tin foil to avoid light exposure. Keep ~1 week.
- It is important to use fresh PFA, as improperly fixed cells can lyse after the addition of MilliQ water.
- Aspirate supernatant and resuspend the cells in 1 mL 1.6% PFA. Allow for staining in dark at room temperature for 5 min.
- Wash cells 2× with 1 mL cold CSM at 400× g for 5 min before aspirating supernatant.
- Resuspend cells in the following:
- 60 uL MACS cleavable anti-biotin beads (20 uL/107 cells) + 30 uL MACS cleavable anti-FITC beads (10 uL/107 cells) + 210 uL CSM (final volume should be 100 uL/107 cells)
- The amount of anti-biotin and anti-FITC beads to add should be determined beforehand using the manufacturer’s instructions.
- Vortex briefly to mix and incubate cells for 15 min at 4 °C in the dark.
- Add 1 mL cold CSM to the mixture and centrifuge at 400× g for 5 min. Aspirate the supernatant.
- During this time, place MACS LS column on MACS magnet. Allow 3 mL of cold CSM to flow through the column to prime the column.
- Resuspend cells in 1 mL CSM and add to LS column. Collect “depleted” flow through in 15 mL conical tube.
- Wash unbound cells from the column by adding 3 mL cold CSM on top of the LS column and allowing the volume to completely flow through the column into the depleted fraction. Repeat with another 3 mL of cold CSM.
- Place a new 15 mL conical tube labeled “enriched” under the LS column and fill with 6 mL of cold CSM. Plunge the CSM through the column using equal steady pressure to collect cells held in the magnetic field using the provided plunger.
- Centrifuge both depleted and enriched fractions of cells at 400× g for 10 min at 4 °C.
- Resuspend the depleted fraction in an appropriate volume to re-count. Resuspend 2–3 × 106 cells from the depleted fraction in 1 mL cold CSM in a 1.5 mL Eppendorf tube. Resuspend the enriched fraction in 1 mL cold CSM in a 1.5 mL Eppendorf tube (we assume the number of cells in our enriched fraction is 1 million cells).
- Add 20 uL multi-sort release agent per 1 mL cell suspension. Vortex briefly to mix and incubate for 10 min at 4 °C in the dark.
- Wash cells by adding 9–10 mL cold CSM to the cell suspension and centrifuge at 400× g for 10 min at 4 °C.
- Aspirate supernatant and wash again in 1 mL cold CSM at 400× g for 10 min at 4 °C.
- Partially aspirate supernatant leaving approximately 50 uL of CSM in the tube.
- Surface Staining
- Create 100 uL 2X antibody master mix (MM) of surface antibodies from Table 2 in CSM.
- Note: The optimal amount of each metal labeled antibody to add should be determined beforehand.
- Add 2 uL each of 160Gd-anti-FITC and 170Er-anti-biotin antibodies to the MM to detect tetanus and insulin-reactive B cells, respectively.
- Add 50 uL of MM to the depleted sample and 50 uL MM to the enriched sample, such that the final concentration of antibodies per sample is 1X. Vortex briefly and incubate on ice for 15–20 min in dark.
- Add 1 mL CSM to samples and centrifuge at 400× g for 5 min.
- Aspirate supernatant and resuspend in 100 uL of 1.6% PFA. Allow cells to stain in dark for 5 min. Add 1 mL CSM and centrifuge at 400× g for 5 min.
- While spinning, create an intercalator cocktail:
- 1 mL 1.6% PFA + 0.5 uL intercalator IR per sample.
- Add 1 mL of intercalator solution to each sample and leave at 4 °C for a minimum of 20 min or up to 1 week.
- Wash 3× in MilliQ water.
- Resuspend in 1 mL MilliQ water per million cells for running on mass cytometer.
- Data acquisition and analysis
- Acquire data on mass cytometer, as previously described [21].
- Analyze data using third-party analysis software, such as FlowJo (Treestar) or CellEngine [22].
3. Representative Results
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Name: | Company: | Catalog Number: | Comments: |
---|---|---|---|
LS Columns | Miltenyi Biotech | 130-042-401 | |
Anti-Biotin Multi-sort Kit | Miltenyi Biotech | 130-091-256 | |
Anti-FITC Multi-sort Kit | Miltenyi Biotech | 130-058-701 | |
MACS manual separators | Miltenyi Biotech | Variable | |
FcR Blocking Reagent human | Miltenyi Biotech | 130-059-901 | |
Tetanus-toxoid | Colorado Serum Company | Unadjuvanted with no carrier proteins present | |
Human Insulin Biotinylated | Eagle Biosciences | INS30-G100 | |
PBS without calcium and magnesium | No contact with autoclaved bottles or beakers or dish soap | ||
Ficoll-Paque PLUS | VWR International | 95021-205 | |
Pierce™ FITC Antibody Labeling Kit | ThermoFisher Scientific | 53027 | |
Pierce™ 16% Formaldehyde (w/v), Methanol-free | ThermoFisher Scientific | 28908 | Dilute to 1.6% with CSM. |
Whole blood in heparinized collection tubes | We typically use at least 30 mL of whole blood to enrich our cells of interest | ||
Cell Staining Media (CSM) (PBS + 0.5% BSA + 2 mM EDTA) | No contact with autoclaved bottles or beakers or dish soap | ||
50 mL conical Tubes | |||
15 mL conical Tubes | |||
1.5 mL Eppendorf tubes | |||
MAXPAR-labeled antibodies of interest | |||
160Gd-anti-FITC antibody | Fluidigm | 3160011B | |
170Er-anti-biotin antibody | Fluidigm | 3170003B | |
Cisplatin live/dead | Fluidigm | 201195 | Stock at 25 mM |
DNA Intercalator | Fluidigm | 201192B | 500 uM stock |
Metal Isotopes: | Specificity: | Clone: | Vendor: |
---|---|---|---|
113Cd | CD45 | HI30 | Fluidigm |
115In | CD3 | HIT3a | Biolegend |
141Pr | CD22 | HIB22 | Biolegend |
142Nd | CD19 | HIB19 | Fluidigm |
143Nd | CD5 | UCHT2 | Fluidigm |
144Nd | CD69 | FN50 | Fluidigm |
145Nd | CD95 | DX2 | Biolegend |
146Nd | IgD | IA6-2 | Fluidigm |
147Sm | CD20 | 2H7 | Fluidigm |
148Nd | CD38 | HIT2 | Biolegend |
149Sm | CD24 | ML5 | Biolegned |
150Nd | CD86 | IT2.2 | Fluidigm |
151Eu | HLA-DR | G46-6 | Fluidigm |
152Sm | CD36 | 5-271 | Fluidigm |
153Eu | CD8 | SK1 | Biolegend |
154Sm | CD40 | 5C3 | Biolegend |
155Gd | CD27 | L128 | Fluidigm |
156Gd | CXCR3 | G025H7 | Fluidigm |
158Gd | CD10 | HI10a | Fluidigm |
159Tb | IgKappa | MHK-49 | Biolegend |
160Gd | Anti-FITC | FIT-22 | Fluidigm |
161Dy | CD80 | 2D10.4 | Fluidigm |
162Dy | CD11c | Bu15 | Fluidigm |
163Dy | CD72 | 3F3 | Biolegend |
164Dy | CD71 | CY1G4 | Biolegend |
165Ho | CXCR5 | J252D4 | Biolegend |
166Er | CD23 | B3B4 | Biolegend |
167Er | IgLambda | 1-155-2 | Biolegend |
168Er | CD138 | DL-101 | Fluidigm |
169Tm | IgG | G18-145 | BD Pharmingen |
170Er | Anti-biotin | 1D4-C5 | Fluidigm |
171Yb | PD-L1 | 29E.2A3 | Biolegend |
172Yb | IgM | MHM-88 | Fluidigm |
173Yb | CD21 | Bu32 | Biolegend |
174Yb | CD4 | SK3 | Fluidigm |
175Lu | CCR4 | L291H4 | Fluidigm |
176Yb | PD-1 | EH12.2H7 | Biolegend |
191Ir | Intercalator | Fluidigm | |
193Ir | Intercalator | Fluidigm | |
195Pt | Live-dead | Fluidigm |
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Stensland, Z.C.; Smith, M.J. Enrichment and Detection of Antigen-Binding B Cells for Mass Cytometry. Magnetochemistry 2021, 7, 92. https://0-doi-org.brum.beds.ac.uk/10.3390/magnetochemistry7070092
Stensland ZC, Smith MJ. Enrichment and Detection of Antigen-Binding B Cells for Mass Cytometry. Magnetochemistry. 2021; 7(7):92. https://0-doi-org.brum.beds.ac.uk/10.3390/magnetochemistry7070092
Chicago/Turabian StyleStensland, Zachary C., and Mia J. Smith. 2021. "Enrichment and Detection of Antigen-Binding B Cells for Mass Cytometry" Magnetochemistry 7, no. 7: 92. https://0-doi-org.brum.beds.ac.uk/10.3390/magnetochemistry7070092