1. Introduction

Immunophenotyping is the use of antigen expression for the identification of distinct immune cell subsets (and their activation statuses) [1–4]. This technique can detect minute changes in cell populations and thus is used to characterize the cell makeup in many diseases as well as determine effects of treatments, such as nanoparticles [5]. It is important to develop a method that allows for immunological evaluation of nanoparticles because some nanoparticles are designed to modify the immune system while others cause immunotoxicity [4, 5]. Currently, the most common technique used to perform immunophenotyping is multicolor flow cytometry [2, 3].

NCL protocol ITA-37 covers two separate immunophenotyping panels (with 11-12 antibody-fluorophore conjugates):

• Immunophenotyping Panel #1 (or Lymphocyte Panel):
  This panel includes antibody-fluorophore conjugates that allow for analysis of different lymphocyte populations including B cells and T cells (CD8+ T cells, CD4+ T cells, regulatory T (Treg) cells, naïve T cells, and γδ TCR T cells). This panel also determines cellular CD25 and CD154 expression which are markers of proliferation and co-stimulation/presentation, respectively.
• Immunophenotyping Panel #2 (or Monocyte, Dendritic cell (DC), Natural Killer (NK)Cell Panel):
  This panel includes antibody-fluorophore conjugates that allow for analysis of different cell populations including CD14+ monocytes, DCs (plasmacytoid (p) and myeloid (m) DCs), and NK cells along with NK T cells. This panel also examines cellular CD69 and CD54 expression which are markers of early activation and adhesion, respectively.

When used in conjunction with other immunoassays, this protocol aids in establishing efficacy and safety profiles of engineered nanoparticles used for vaccine or drug delivery. This protocol has two parts, ITA-37.1 described in this document and intended for instrument calibration, and ITA-37.2, described in a separate document and intended for the analysis of nanoparticle-treated cells.

2. Principle

This protocol (ITA-37.1) uses compensation beads and peripheral blood mononuclear cells (PBMC) derived from the blood of healthy donor volunteers for instrument calibration and reagent qualification. Following this protocol is required before proceeding to the second part of the protocol, ITA-37.2, for the immunophenotyping of nanoparticle-treated cells.

Experimental steps described herein, allow for proper optimization and compensation of the antibody-fluorophore conjugates used in the panels. The antibody titrations allow for the optimization of antibody concentrations in which the optimal concentration/ dilution is defined as the concentration that leads to the greatest difference between the positive and negative mean fluorescence intensity. Single stain control experiments performed with compensation beads allow for the necessary compensation of the optimized antibody-fluorophores used in each panel. Lastly, fluorescence minus one (FMO) controls serve as a way to fine-tune the compensation obtained from the single stain control experiment to account for any minute changes in the protocol, including the presence of PBMC rather than compensation beads and fixation.

The variety and complexity of immune cell phenotypes that can be assessed during immunophenotyping depends on the available flow cytometry instrumentation. This protocol is optimized for the flow cytometer NovoCyte 3005 by Acea Biosciences Inc. (part of Agilent Technologies), which has the capacity to acquire data from 14 distinct fluorophores simultaneously. NovoCyte 3005 is equipped with 3 lasers (405 nm, 488 nm, and 640 nm) and 6 detectors (445/45 nm, 530/30 nm, 572/28 nm, 660/20 nm, 725/40 nm and 780/60 nm) [6].

If another cytometer is used, the procedure described herein would require an optimization according to the technical specifications of that instrument.

3. Reagents, Materials, and Equipment

Note: The NCL does not endorse any of the suppliers listed below; these reagents were used in the development of the protocol and their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted. Please note that suppliers may undergo a name change due to a variety of factors. Brands and part numbers typically remain consistent but may also change over time.

3.1.

Reagents for PBMC cultures

3.1.1.

Human blood anti-coagulated with Li-heparin and obtained from healthy donors

3.1.2.

Phosphate buffered saline (PBS) (GE Life Sciences, SH30256.01)

3.1.3.

RPMI-1640 (GE Life Sciences, HyClone, SH30096.01)

3.1.4.

Fetal bovine serum (GE Life Sciences, HyClone, SH30070.03)

3.1.5.

Penicillin streptomycin solution (GE Life Sciences, Hyclone, SV30010)

3.1.6.

L-glutamine (GE Life Sciences, Hyclone, SH30034.01)

3.1.7.

Ficoll Paque Premium (GE Healthcare, 17-5442-03)

3.1.8.

Hank’s balanced salt solution (HBSS) (Gibco, 14175-095)

3.1.9.

ViaStain AOPI Staining solution (Nexcelom Biosciences, CS2-0106-5mL)

3.2.

Controls

3.2.1.

Phytohemagglutinin (PHA-M) (Sigma, L8902)

3.2.2.

Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, P1585)

3.2.3.

Ionomycin (STEMCELL Technologies, 73722)

3.2.4.

Oligodeoxyribonucleotide, Human TLR9 ligand (ODN2216) (InvivoGen, tlrl-2216-1)

3.3.

Reagents for Flow Cytometry

3.3.1.

UltraComp eBeads Plus Compensation Beads (Invitrogen, 01-3333-42)

3.3.2.

eBioscience Flow Cytometry Staining Buffer (Invitrogen, 00-4222-26)

3.3.3.

Paraformaldehyde (PFA) 20% Solution (Electron Microscopy Science, 15713)

3.3.4.

NovoRinse (Agilent Technologies, 872B603)

3.3.5.

NovoClean (Agilent Technologies, 872B602)

3.3.6.

NovoFlow (Agilent Technologies, 871B607)

3.3.7.

Antibodies/Dyes (Tables 2–3)

3.4.

Equipment and Materials

3.4.1.

Pipettes covering a range of 0.05 to 10 mL

3.4.2.

96-well U-bottom plates

3.4.3.

24-well round bottom plates

3.4.4.

Polypropylene tubes, 15 and 50 mL

3.4.5.

Microcentrifuge tubes

3.4.6.

5 mL Polystyrene round bottom tubes; 12×75 mm

3.4.7.

Centrifuge

3.4.8.

Refrigerator, 2-8 °C

3.4.9.

Freezer, −20 °C

3.4.10.

Cell culture incubator with 5% CO₂ and 95% humidity

3.4.11.

Biohazard safety cabinet approved for level II handling of biological material

3.4.12.

Water bath

3.4.13.

Vortex

3.4.14.

Cellometer Auto2000

3.4.15.

Acea Novocyte 3005

Table 1

Guidance on Concentrations and Purpose of Positive Controls.

Table 2

Anti-human Labeling Antibodies used in the Immunophenotyping Panel 1 (Lymphocyte Panel).

Table 3

Anti-human Labeling Antibodies for Immunophenotyping Panel 2 (Monocyte, DC, NK Cell Panel).

4. Preparation of Reagents and Controls

4.1.

Reagent and Control Preparation for PBMC

4.1.1.

Complete RPMI-1640 medium

The complete RPMI medium should contain the following reagents:

10% FBS (heat inactivated)

2 mM L-glutamine

100 U/mL penicillin

100 μg/mL streptomycin

Store at 2-8 °C protected from light for no longer than 1 month. Before use, warm the media in a water bath.

4.1.2.

Heat inactivated fetal bovine serum

Thaw a 50 mL aliquot of fetal bovine serum and equilibrate to room temperature. Place the tube in a water bath set up to 56 °C and incubate with mixing for 35 min. The heat inactivation takes 30 min and the initial 5 min is used to bring the entire content of the vial to 56 °C. Chill the serum and use to prepare complete culture media.

4.1.3.

PMA (1 mg/mL stock)

Reconstitute in DMSO to a final concentration of 1 mg/mL. Prepare single use 20 μL aliquots and store at −20 °C. On the day of experiment thaw an aliquot at room temperature and dilute in culture media so the final concentration in the test sample is 50 ng/mL.

4.1.4.

Ionomycin (10 mg/mL stock)

Ionomycin is supplied in ionomycin free acid 1%, ethyl alcohol 99%. Prepare single use 5 μL aliquots and store at −20 °C. On the day of experiment thaw an aliquot at room temperature and dilute in culture media so the final concentration in the test sample is 1 μg/mL.

4.1.5.

ODN 2216 (1 mg/mL stock)

This oligonucleotide is supplied as lyophilized powder. Reconstitute in pyrogen-free, nuclease-free water to a final concentration of 1 mg/mL. Prepare single use 5 μL aliquots and store at −20°C. On the day of experiment thaw an aliquot at room temperature and dilute in culture media so the final concentration in the test sample is 5 μg/mL.

4.1.6.

Phytohemagglutinin (PHA-M, 1 mg/mL stock)

Add 1 mL of sterile PBS or cell culture medium per 1 mg of PHA-M to the vial and gently rotate to mix. Store daily use aliquots at a nominal temperature of - 20°C. Avoid repeated freezing/thawing. On the day of experiment dilute stock PHA-M solution in cell culture medium so the final concentration in the positive control sample is 10 μg/mL.

4.2.

Collection and Handling of Whole Blood

This step requires approval by an Institutional Review Board (IRB) or another relevant board; please consult your organization for details on appropriate regulations within your research organization. Collect whole blood from healthy donor volunteers who have not been on medication and clear from infection for at least 2 weeks prior to the blood donation. Use Li-heparin tubes and discard first 10 cc. For best results, whole blood should be used within 1 hour after collection. Prolonged storage (> 2 hr) of whole blood will lead to a decrease in cell function.

4.3.

Preparation of PBMC

Note: PBMC isolated during this step are used in steps 6 (specifically, live: dead cell calibration described in 6.1.1.3 and 6.2.1.3), and 7 of this protocol. Procedures described in steps 6 and 7 may be performed on a separate day using PBMCs freshly isolated that day.

4.3.1.

Place freshly drawn blood into 15- or 50-mL conical centrifuge tubes, add an equal volume of room-temperature PBS, and mix well.

4.3.2.

Slowly layer the Ficoll-Paque solution underneath the blood/PBS mixture by placing the tip of the pipet containing Ficoll-Paque at the bottom of the blood sample tube. Alternatively, the blood/PBS mixture may be slowly layered over the Ficoll-Paque solution. Use 3 mL of Ficoll-Paque solution per 4 mL of blood/PBS mixture. For example, 15 mL Ficoll-Paque per 20 mL of diluted blood in a 50 mL tube.

Note: To maintain Ficoll-blood interface it is helpful to hold the tube at a 45° angle.

4.3.3.

Centrifuge 30 min at 900 x g, 18-20°C, without brake.

Note: Depending on the type of centrifuge, one may also need to set acceleration speed to minimum.

4.3.4.

Using a sterile pipet, remove the upper layer containing plasma and platelets and discard it.

4.3.5.

Using a fresh sterile pipet, transfer the mononuclear cell layer into another centrifuge tube.

4.3.6.

Wash cells by adding an excess of HBSS and centrifuging for 10 min at 400xg, 18-20 °C. The HBSS volume should be ~3 times the volume of mononuclear layer.

Note: Usually 4 mL of blood/PBS mixture results in ~ 2 mL of mononuclear layer and requires at least 6 mL of HBSS for the wash step. We use 10 mL of HBSS per each 2 mL of cells.

4.3.7.

Discard supernatant and repeat wash step one more time.

4.3.8.

Re-suspend cells in complete RPMI-1640 medium. Dilute cells 1:2 with AOPI, count cells and determine viability using AOPI exclusion. If viability is at least 90%, proceed to step 7.1.1.

Note: The cells isolated during this step can also be used to prepare live: dead cell controls for steps 6.1.1.3 and 6.2.1.3.

5. NovoCyte 3005 Instrument Settings

Important Note: The following experiments were optimized on the NovoCyte 3005 with the settings shown in Figure 1. If a different cytometer is used, then instrument calibration or fluorescent labels may need to be changed or adjusted accordingly.

Figure 1

Settings on NovoCyte 3005 [7].

6. Flow Cytometry Control Experiments with Compensation Beads for Antibody Titration and Single Stain Controls

Important Note: The following procedures need to be completed for both Immunophenotyping Panel #1 and #2.

6.1.

Antibody Titration (Optional)

Prepare a 6-point concentration curve that includes the antibody at the stock concentration (between 50 μg/mL to 400 μg/mL for a given antibody) and five 5-fold serial dilutions (dils) of the stock (Dils 5; 25; 125; 625; and 3125). Please check with manufacturer to determine that the concentration for a given antibody remains unchanged.

Note: Antibody titration needs to be performed to establish the antibody/dye dilutions for the panels that will be run. Please refer to Tables 2–3 for the antibody-fluorophore combinations used for Immunophenotyping Panels #1 and #2, respectively. Once the panel is established, the antibody titrations do not need to be run again unless there are issues encountered with a new antibody lot or the stock concentration changed. Antibody dilutions have already been determined for panels used herein.

6.1.1.

Bead and Live: Dead PBMC Plate Preparation (without antibodies or dyes)

6.1.1.1.

Make a 10-fold dilution of UltraComp eBeads Plus Compensation Beads using staining buffer as the diluent (making sure to vortex beads).

6.1.1.2.

Add 50 μL of 10-fold diluted beads to each of the necessary 96-well U-bottom wells of the plate labeled “Bead and Live: Dead PBMC Plate” (6 wells of beads needed per antibody). (Please see Tables 2–3 for antibodies used in Immunophenotyping Panels #1 and #2 and Plate 1 in the Appendix for an example of plate setup.)

6.1.1.3.

Add 50 μL of PBMC suspended in 1X PBS to each of the necessary wells of the 96-well plate labeled “Bead and Live: Dead PBMC Plate” (6 wells per dye). Ideally, this sample should contain 1:1 mixture of live and dead PBMC at a total concentration of approximately 5×10⁵ cell/mL. See Plate 1 setup in the Appendix for an example.

Note: Freshly isolated or freeze thawed PBMC work well. If necessary, cells can be heat shocked at 70°C to obtain about 50% cell death.

6.1.2.

Antibody/Dye Titration Plate Preparation

6.1.2.1.

Add 20 μL of staining buffer to each appropriate well in a 96-well U bottom plate labeled “Antibody/Dye Titration Plate” for preparation of the antibody dils (5 wells in a column prepared for each antibody—Wells B-F). Please see Plate 2 in Appendix for an example.

6.1.2.2.

Add 20 μL of 1X PBS to each appropriate well in the 96-well U bottom plate labeled “Antibody/Dye Titration Plate” for preparation of the Zombie Aqua dye dils (5 wells in a column—Wells B-F). Please see Plate 2 in Appendix for an example.

6.1.2.3.

Prepare each antibody/dye titration down a single column of the plate as follows:

1. Add 5 μL of appropriate antibody/dye to well B1; mix well.
2. Transfer 5 μL of diluted antibody/dye from B1 and add it to well C1; mix well.
3. Transfer 5 μL of diluted antibody/dye from C1 and add it to well D1; mix well.
4. Transfer 5 μL of diluted antibody/dye from D1 and add it to well E1; mix well.
5. Transfer 5 μL of diluted antibody/dye from E1 and add it to well F1; mix well.
6. Repeat steps a–e for each antibody in appropriate wells.
   Please see Plate 2 in Appendix for an example.

6.1.3.

Combination of Antibody/Dye Titrations to Bead and Live: Dead PBMC Plate

6.1.3.1.

Add 5 μL of appropriate stock antibody/dye to corresponding well in row A of the “Bead and Live: Dead PBMC Plate.”

6.1.3.2.

Transfer 5 μL of appropriate antibody/dye dilution from “Antibody/Dye Titration Plate” to corresponding well of the “Bead and Live: Dead PBMC Plate.”

Note: Another ~10 fold-dilution applied to each dilution from the titration (5 μL antibody into 50 μL diluent) that will be taken into account in subsequent calculations.

6.1.3.3.

Incubate the “Bead and Live: Dead PBMC Plate” (now with beads + antibody and PBMC + dye) in the dark at room temperature for 30 minutes.

6.1.3.4.

Centrifuge plate for 1-5 minutes at 300xg.

6.1.3.5.

Manually aspirate 40 μL of supernatant from each well.

6.1.3.6.

Add 40 μL of staining buffer to each well. Resuspend pellets.

6.1.3.7.

Plate ready to read.

6.1.4.

Plate Reading with NovoCyte 3005

6.1.4.1.

Turn on computer > NovoExpress > username and password

6.1.4.2.

Turn on flow cytometer

6.1.4.3.

Check default parameters: all parameters

6.1.4.4.

Set up the experimental parameters (see Table 4.)

1. Plate Manager

   1. Mode: custom; 96 well u-bottom
   2. Click on appropriate squares to create sample. Rename each well accordingly in work list.
   3. Indicate to run samples vertically.
   4. Mix every 12 wells; 1500 rpm, 10sec; No rinse
2. Experimental Run. Stop conditions: Events 12,000; 30 μL. Flow rate: Fast.
3. Cytometer Settings: Parameters, see Table 4.

6.1.4.5.

Insert plate.

6.1.4.6.

Click run plate. Highlight all boxes that are to be run (all). Click run; Click okay

6.1.4.7.

Save file.

6.1.5.

Data Analysis in NovoExpress

6.1.5.1.

Make a forward scatter (FSC) vs side scatter (SSC) plot. Draw an oval region of interest (ROI) around the beads.

6.1.5.2.

Make a histogram by double-clicking on the bead population. Change the axes of the histogram (right click). X-axis: appropriate fluorophore; Y-axis count. Drag the analysis tab to each sample of corresponding fluorophore.

6.1.5.3.

To overlay graphs, duplicate histogram by right clicking. Right click on the duplicated histogram → Edit overlay → Add → Select the diluted samples to add → Add and close

6.1.5.4.

Right click on graph → style → partial overlap

6.1.5.5.

Copy image and make a PowerPoint with serial dilution.

6.1.5.6.

Optimal dilution is determined by the antibody/dye concentration that has the largest difference between positive and negative stain. > 2 log differences needed. Need negative to be as low as possible without large shift in positive population.

6.1.5.7.

Repeat analysis for each serial dilution.

6.1.5.8.

Save files.

6.2.

Single Stain Controls with Compensation Beads

Important Note: Must be completed each time a new antibody lot is obtained. One replicate per condition. Please see Tables 2–3 for antibody-fluorophore combinations used in single stain controls for Immunophenotyping Panels #1 and #2, respectively.

6.2.1.

Plate Preparation with Beads and Cells

6.2.1.1.

Make a 10-fold dilution of UltraComp eBeads Plus Compensation Beads using staining buffer as the diluent (making sure to vortex beads).

6.2.1.2.

Add 50 μL of diluted beads to appropriate wells of a 96-well U-bottom plate (1 well/antibody).

6.2.1.3.

Add 50 μL of cells prepared in 1X PBS (1:1 live: dead PBMC; approximately 5×10⁵ cell/mL) to 2 wells. At the end of this step, one should have 2 wells total: 1 well prepared for Zombie Aqua dye and 1 well prepared for unstained PBMC.

Note: Freshly isolated or freeze thawed PBMC work well. If necessary, cells can be heat shocked at 70°C for 2 hours to obtain about 50% cell death.

6.2.2.

Antibody Dilution Preparation

6.2.2.1.

Prepare antibody dilutions (at least 5 μL each) with staining buffer as previously determined by antibody titration (Step 6.1) and indicated in Table 5.

Important note: Zombie Aqua dye dilution is to be prepared in 1X PBS.

6.2.2.2.

Transfer 5 μL of antibody dilution/dye to corresponding well of the plate with the beads/cells.

6.2.2.3.

Incubate plate in the dark for about 30 minutes.

6.2.2.4.

Centrifuge plate for 5 min at 300xg.

6.2.2.5.

Manually aspirate 30 μL of supernatant from the well.

6.2.2.6.

Add 50 μL of staining buffer to the well and resuspend pellet.

6.2.2.7.

Plate ready to read on the cytometer.

6.2.3.

Plate Reading with NovoCyte 3005

6.2.3.1.

Turn on computer > NovoExpress > username and password

6.2.3.2.

Turn on flow cytometer

6.2.3.3.

Save file.

6.2.3.4.

Instrument → Auto compensation

6.2.3.5.

In pop-up, de-select unused parameters. Rename other channels to the fluorophores used → Okay

Note: Panel #1 de-select Area and Height Parameters for PE-Cy5 and PerCP-eFluor710; Panel #2 de-select PE-Cy5, PerCP-eFluor710, and QDot705

6.2.3.6.

Set up the experimental parameters

1. Plate Manager

   1. Mode: custom; 96 well u-bottom
   2. Select wells and rename and reposition each well accordingly in work list.
   3. Indicate to run samples vertically.
   4. Mix every 2 wells; 1500 rpm, 10sec; Rinse every 3 wells
2. Experimental Run. Stop conditions: Events 25,000; 50 μL. Flow rate: Fast.
3. Cytometer settings: Parameters, please see Table 4 in Section 6.1.4.4.

   Note: Need 2500 positive events for software to compensate correctly.

6.2.3.7.

Insert plate into flow cytometer.

6.2.3.8.

Click run plate. Highlight all boxes that are to be run (all) → run → Okay

6.2.3.9.

Can adjust main cell gate for cell samples.

6.2.3.10.

Save file.

6.2.3.11.

Export FCS files and save compensation matrix.

Table 4

Cytometer Parameters. Left: Panel #1; Right: Panel #2 Note: After samples are created, make sure that the experimental settings for each specimen are the same. Whenever a change is made, apply change to all and save file.

Table 5

Human Immunophenotyping Panel Antibody Dilutions.

7. Flow Cytometry Control Experiments with PBMC (FMO Controls) (2 Day procedure per panel)

Important Note: This step must be completed each time a new antibody lot is obtained. Please see Tables 2–3 for antibody-fluorophore combinations used in single stain controls for Immunophenotyping Panels #1 and #2, respectively. An unstained sample and a fully stained labeling antibody sample with all antibodies/dyes are also required. One replicate per condition. FMOs for panels may be processed concurrently or separately.

7.1.

Experimental procedure for PBMC (Day 1)

Note: This procedure is optimized for 24 well plate. If other plates are used, the volumes may need to be adjusted accordingly.

7.1.1.

Adjust concentration of PBMC from step 4.3.8 to 1.25 × 10⁶ viable cells/mL using complete RPMI medium.

7.1.2.

Dispense 800μL of PBMC per well in 24 well plate. Gently shake plates to allow all components to mix.

7.1.3.

Dispense 200 µL of positive control samples into corresponding wells of 24 well plate containing 800 µL of PBMC for a total of 1 mL per well.

1. Immunophenotyping Panel #1: Positive Control = PMA/Ionomycin
2. Immunophenotyping Panel #2: Positive Control = ODN2216/PHA-M

7.1.4.

Incubate for about 24 hours in a humidified 37°C, 5% CO₂ incubator.

7.2.

Experimental PBMC staining procedure (FMO controls) (Day 2)

7.2.1.

Heat Shocked PBMC Preparation

7.2.1.1.

Obtain needed quantity of treated PBMC from the 24-well plate and transfer to a 15 mL conical tube.

Note: 0.25×10⁶ heat shocked PBMC needed per sample; rest of PBMC will be used in step 7.2.2.1.

7.2.1.2.

Centrifuge sample for 10 min at 400xg. Aspirate the supernatant and resuspend the pellet in 1 mL 1X PBS.

7.2.1.3.

Transfer sample to microcentrifuge tube and place sample in digital heat block set to 70°C for at least 1.5 hours.

7.2.2.

PBMC Preparation

7.2.2.1.

Transfer 750 μL of cell sample (0.75×10⁶ cells/well) to appropriately labeled microcentrifuge tubes.

Note: Samples needed = unstained control, FMO control for each labeling antibody, fully stained control containing all labeling antibodies/dyes

7.2.2.2.

Centrifuge samples at 400xg for 7 min. Aspirate each supernatant and resuspend each pellet in 1 mL 1X PBS.

7.2.2.3.

Add 62.5 μL heat shocked PBMC (0.25×10⁶ dead PBMC) to each sample.

Note: Total of 1×10⁶ PBMC in each sample (~0.75×10⁶ live cells and ~0.25×10⁶ dead cells)

7.2.3.

Staining Procedure

7.2.3.1.

Centrifuge each sample at 400xg for 7 min. Aspirate each supernatant and resuspend each pellet in 49.2 μL of 1X PBS.

7.2.3.2.

Make 5-fold dilution of Zombie Aqua dye for staining samples with 1X PBS as the diluent.

Note: One will need 0.8 μL of 5-fold diluted Zombie Aqua dye x sample number (n) = 0.8 (n+2) μL of the 5-fold dilution.

7.2.3.3.

Add 0.8 μL of a 5-fold diluted Zombie Aqua dye to all samples except unstained sample and Zombie Aqua FMO sample.

7.2.3.4.

Incubate samples for 30 minutes at room temperature in the dark.

7.2.3.5.

Wash each sample 2x with 500 μL staining buffer.

1. Add 500 μL staining buffer to each tube.
2. Centrifuge samples for 7 min at 400xg.
3. Aspirate off each supernatant.

7.2.3.6.

Resuspend each sample in 40 μL staining buffer.

7.2.3.7.

Prepare 5-fold antibody dilutions in staining buffer in labeled microcentrifuge tubes (one dilution per antibody needed; see Table 6).

7.2.3.8.

Make a master mix (MM) for each FMO control in an appropriately labeled microcentrifuge tube.

Note: Add 5-fold dilution antibodies to the appropriate microcentrifuge tube as indicated in Tables 7–8 (Single column indicates a single tube). Add necessary amount of staining buffer to make final volume 60 μL.

7.2.3.9.

Add appropriate FMO MM (60 μL) to appropriate cell sample (40 μL) (prepared in step 7.2.3.6).

7.2.3.10.

Incubate samples for 30 min at room temperature in the dark.

7.2.3.11.

Wash each sample 2x with 500 μL staining buffer.

7.2.3.12.

Fix cells by resuspending each cell sample in 100 μL 2% PFA and incubating for 15 minutes at room temperature in the dark.

7.2.3.13.

Wash each sample 2x with 500 μL staining buffer.

7.2.3.14.

Resuspend each sample in 500 μL staining buffer and transferred samples to appropriately labeled flow tubes.

7.2.3.15.

Briefly vortex samples.

Table 6

Volumes of 5-fold Antibody Dilutions Needed for FMOs.

Note: Prepare enough diluted antibodies for n + 2. Each antibody is only needed at one previously determined optimal dilution (see Table 5). I.e., for Immunophenotyping Panel #1, the antibody dilution that was determined to lead to the optimal concentration of CD8-FITC was Dil 25; therefore, 8 μL of the 5-fold dilution of CD8-FITC is needed per FMO control.

Table 7

Immunophenotyping Panel #1 FMO Control Samples.

Table 8

Immunophenotyping Panel #2 FMO Control Samples.

7.3.

Data Acquisition with NovoCyte 3005 for FMO controls

7.3.1.

Turn on computer > NovoExpress > username and password

7.3.2.

Turn on flow cytometer.

7.3.3.

Save file.

7.3.4.

Check the experimental parameters.

1. Plate Manager

   1. Mode: custom; 24 tube rack
   2. Indicate samples to run horizontal.
   3. Click on proper squares to create sample. Rename each sample accordingly in work list.
   4. Mix: 1 cycle every, 2 wells (1500 rpm; 10 sec); Rinse: 1 cycle, every 3 wells
2. Experimental Run. Stop conditions: Events 300,000; 300 μL. Fast (Apply changes to all samples)
3. Cytometer Settings: Parameters, please see Table 4 in Section 6.1.4.4. (Make sure channels and laser intensity are the same as the single stain control experiment.)

7.3.5.

Insert samples in tube rack.

7.3.6.

Click run plate. Highlight all boxes that are to be run (all). → run → okay

7.3.7.

Save file.

7.3.8.

Export FCS files.

7.4.

Data Analysis of FMO controls with NovoExpress

Overview: The necessary data from this analysis is the compensation/spillover matrices. The FMO control samples need to be compensated with the compensation matrix from the single stain control samples. The compensation matrix may then need to be adjusted to account for the differences between beads (single stain controls) and cells (FMO controls).

7.4.1.

Application of compensation matrix to FMO controls

Note: There are multiple ways to apply the compensation matrix of the single stain controls to the FMO controls. Below is one method.

7.4.1.1.

Make a copy of the single stain control file (.ncf) that correlates to the FMO control file.

7.4.1.2.

Upload the FCS files from the appropriate FMO control experiment into the single stain control file (.ncf).

1. Right click on file name in Experimental Manager.
2. Click on Import FCS files.
3. Browse for the folder with appropriate FCS files.

7.4.1.3.

Apply the compensation matrix from the single stain controls to the FMO samples.

1. Under “Compensation Specimen” click on the “+” for any sample.
2. Right click on Compensation and select Copy.
3. Right click on overarching FMO control experimental sample and click on Paste.
4. “Are you sure you want to paste Compensation to all samples in experimental sample name?” Yes.
5. Can then delete the single stain Compensation Specimen.

7.4.1.4.

Rename the file with the compensated FMO samples and save.

7.4.2.

Analysis Method/Compensation Adjustment of FMO controls

7.4.2.1.

Make plots on each FMO sample; Refer to Figure 2 for examples of gates

1. Make a plot of FSC-H vs. SSC-H and gate main the cell population “P1”.
2. Make a plot of FSC-A vs. FSC-H from “P1” and gate the single cells “P2”.

7.4.2.2.

Adjust compensation using the fully stained sample.

1. Make a plot of FSC-H vs. SSC-H and gate main the cell population “P1”.
2. Make a plot of FSC-A vs. FSC-H from “P1” and gate the single cells “P2”.
3. Make a plot of each fluorophore pair (example FITC vs. PE; FITC vs. PE-Cy-7; FITC vs. APC; etc.) from “P2”.

   Note: Plots can auto-populate by clicking on the dot plot symbol until all pairwise fluorophore plots are plotted.

4. Right click on pairwise plots and click Edit overlay. Add FMO sample that corresponds to the fluorophore on the x-axis. Apply.
5. For plots that have large compensation, check the compensation. Adjust the compensation of the parameters using the quick compensation adjustments on the fully stained sample. (Adjust plots to eliminate or minimize the slanting in the FMO overlay)
6. Once compensation of the fully stained sample is complete, apply the compensation to all the FMO samples.

7.4.2.3.

Save compensated file and export compensated FCS files.

Figure 2

Gating FMO samples. Left: Gate of the cell population; Right: Gate of the singlet population.

8. References

1.

Oughton, J.A. and Kerkvliet, N.I. Immune cell phenotyping using flow cytometry. Current protocols in toxicology. 2005, 23(1), 18.18. 11–18.18. 24. [PubMed: 23045120]

2.

McCoy, J., J Philip, Immunophenotyping. 2019: Springer.

3.

McKinnon, K.M. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018, 120, 5 1 1–5 1 11. DOI: 10.1002/cpim.40. PMID: 29512141. [PMC free article: PMC5939936] [PubMed: 29512141] [CrossRef]

4.

Newton, H.S. and Dobrovolskaia, M.A. Immunophenotyping: Analytical approaches and role in preclinical development of nanomedicines. Adv Drug Deliv Rev. 2022, 185, 114281. DOI: 10.1016/j.addr.2022.114281. PMID: 35405297. [PMC free article: PMC9164149] [PubMed: 35405297] [CrossRef]

5.

Dobrovolskaia, M.A. and McNeil, S.E. Immunological properties of engineered nanomaterials. Nat Nanotechnol. 2007, 2(8), 469–478. DOI: 10.1038/nnano.2007.223. PMID: 18654343. [PubMed: 18654343] [CrossRef]

6.

Acea BioScience Inc., NovoCyte® Flow Cytometer Technical Specifications. 2017.

7.

Agilent Technologies, NovoCyte 3 Lasers 3005 Model 3005-default configuration. 2022.

9. Abbreviations

PBMC

peripheral blood mononuclear cells

Treg

regulatory T cells

DC

dendritic cell

NK

natural killer

pDC

plasmacytoid DC

mDC

myeloid DC

FMO

fluorescence minus one

FBS

fetal bovine serum

PBS

phosphate buffered saline

PHA-M

phytohemagglutinin

ODN

oligodeoxyribonucleotide

PFA

paraformaldehyde

QC

quality control

FSC

forward scatter

SSC

side scatter

ROI

region of interest

MM

master mix

Ab

antibody

10. Appendix

Plate 1Bead and Live: Dead PBMC Plate, Panel #1 Example

Plate 2Antibody/Dye Titration Plate, Panel #1 Example

This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.

*

Address correspondence to: vog.hin.liam@aniram

Newton HS, Zhang, J, Dobrovolskaia MA, NCL Method ITA-37.1: Immunophenotyping: Instrument Calibration and Reagent Qualification for Immunophenotyping Analysis of Human Peripheral Blood Mononuclear Cell Cultures. https://www.cancer.gov/nano/research/ncl/protocols-capabilities