Materials and Reagents

Acquisition of human PBMCs from whole blood

Leucosep Tube, 50 ml (Greiner Bio-One, catalog number: 227290 )
2 ml Cryovial (Starstedt, catalog number: 72.694.006 )
Total peripheral blood (~25 ml) from a healthy donor or patient. In general, 1 ml of blood contains 1 × 10⁶PBMCs.
Ficoll-Paque^TM Plus (GE Healthcare, catalog number: 17-1440-03 )
Fetal bovine serum (FBS, Gibco, catalog number: 10270-106 )
CRITICAL: Heat-inactivate for 1 h at 56 °C prior to use.
20% dimethyl sulfoxide (DMSO, Sigma-Aldrich, catalog number: D2650-100ML)
CAUTION: DMSO is a toxic product and should be handled with care and discarded.
RPMI 1640 Medium containing L-glutamine (Lonza, catalog number: BE12-702F ) supplemented with 5% (vol/vol) fetal bovine serum (Gibco, catalog number: 10270-106 ) (see Recipes)
Freezing medium containing 80% FBS (Gibco, catalog number: 10270-106 ) (see Recipes)

Collection of Murine organs

10 ml syringes (Braun, catalog number: 4616103V ) and 25-gauge × ⅝ inch needle (Braun, catalog number: 4657853 )
C57BL/6J mice (Charles River) that are 8-12 weeks of age, either males and females
Note: All animal experiments should comply with national laws and institutional regulations.
PBS (Gibco, catalog number: 14190169 )

Preparation of Murine organs

100 µm nylon strainers (Corning, catalog number: 352360 )
2 ml syringes (BD Biosciences, catalog number: 307727 )
Nunc Cell Culture/Petri dishes (Thermo Scientific, catalog number: 1533066 )
RPMI 1640 Medium, Glutamax^TM Supplement (Gibco, catalog number: 61870036 ) containing 2% (vol/vol) FBS (Capricorn, catalog number: FBS-12A ) (see Recipes)
CRITICAL: Heat-inactivate for 1 h at 56°C prior to use.

In vitro Stimulation of Single-Cell Suspension/Direct fixation for ex vivo (basal) measurements

96-wells plate (U-shaped, 310 µl/w, Greiner Bio-One, catalog number: 650201 )
eBioscience FoxP3/Transcription Factor staining kit (Invitrogen, catalog number: 00-5523-00 ) containing:
1. Fixation and permeabilization concentrate
  CAUTION: This product contains paraformaldehyde (PFA), a toxic and mutagenic product. It should be handled with care and discarded as hazardous waste.
2. Diluent
3. 10× Wash Buffer (dilute 10 times in deionized water)
Stimuli for BCR signaling and co-receptor engagement, see Table 1.
CAUTION: Sodium azide is highly toxic and dangerous to the environment. It should be handled with care and discarded as hazardous waste.

Table 1. BCR and co-receptor ligands for stimulation of human and murine B cells

Flow Cytometry Procedures

Insert tubes (Greiner Bio-One, catalog number: 102280 ) and 5 ml round bottom polystyrene tubes (Corning, catalog number: 352052 )
UltraComp eBeads^TM Compensation Beads (Invitrogen, catalog number: 01-2222-42 )
10× Wash Buffer (dilute 10 times in deionized water) from the eBioscience FoxP3/Transcription Factor staining kit (Invitrogen)
FcR Blocking agent (Human TruStain FcX, Biolegend, catalog number: 422302 )
Antibodies for flow cytometry are listed in Table 2 (phospho-protein-specific antibodies) and Table 3 (antibodies used for an accurate identification of B cell subpopulations). For mouse studies, another 2.4G2 antibody was used from own production.
CAUTION: Sodium azide is highly toxic (see above).
MACS-buffer containing 0.5% (vol/vol) bovine serum albumin (BSA, Sigma-Aldrich, catalog number: A8327 ) and 2 mM ethylenediaminetetraacetic acid solution (EDTA, Sigma-Aldrich, catalog number: 0 3690 ) in PBS (see Recipes)

Table 2. Phospho-specific antibodies for the analysis of human and mouse B cells and directions on stimulation timing and antibody dilution

Table 3. Antibodies used for accurate gating of B cell subsets

Equipment

Standard laboratory materials or equipment, such as pipettes, tubes, and tips
Tools suitable for sacrificing mice and harvesting organs, including: scissors, tweezers and 70% ethanol
Mortar and pestle
Stopwatch or timer
Water bath incubator and/or CO₂ incubator
Multifuge x 3R Refrigerated Centrifuge (Thermo Scientific, catalog number: 75004515 ) with TX-1000 swinging bucket rotor (Thermo Scientific, catalog number: 75003017 ).
LSR II flow cytometer (BD Biosciences, catalog number: BD LSR-II ) equipped with a red, blue and violet laser
Flow cabinet
4 °C fridge
-80 °C freezer

Software

Data acquisition by FACSDiva^TM Software (BD Biosciences)
FlowJo software (BD Biosciences) to analyze data

Procedure

Sample collection
Acquisition of human PBMCs from whole blood (Timing: 60 min)
Note: Steps 1 to 6 are performed in a sterile environment using a flow cabinet.
1. Add 15 ml of Ficoll to the Leucosep tube and spin down at 1,000 × g for 1 min at room temperature (RT).
2. Gently load a maximum of 25 ml whole blood per Leucosep tube.
  CRITICAL: Make sure the Ficoll is beneath the filter in the tube and that blood does not mix with the Ficoll.
3. Spin down the Leucosep tube containing whole blood and Ficoll at 1,000 × g for 10 min at RT.
  CRITICAL: Set the deceleration as low as possible to keep the Ficoll gradient separation of the PBMC layer intact.
4. Transfer the whole solution of peripheral blood mononuclear cells (PBMCs), Ficoll and Plasma to a 50 ml tube containing 15 ml RPMI 1640 supplemented with 5% FBS.
  CRITICAL: The erythrocyte fraction below the filter should be retained in the Leucosep tube.
5. Spin down the 50 ml tube at 400 × g for 7 min at 4 °C. Afterwards, remove the supernatant and resuspend in 1-5 ml of cold (4 °C) RPMI-2% FBS.
  OPTIONAL: When performing phosphoflow on fresh PBMCs (non-frozen samples), proceed to Step 9.
6. Count cells using standard procedures (manual or automated) and store not more than 50 × 10⁶ viable cells per vial by adding Freezing medium in 1:1 ratio to the volume in which the cells are held. Add 0.5 ml of Freezing medium to 0.5 ml of medium containing 20 × 10⁶- 50 × 10⁶ cells. Transfer the cells into 2 ml cryovials that are suitable for freezing and immediately freeze the vials at a rate of 1 °C per minute.
  CRITICAL: As DMSO is harmful to cells, slowly add the freezing medium to the cells in RMPI 5% FBS while gently shaking the tube.
  PAUSE POINT: Cells can be kept at -80 °C for several weeks or in liquid nitrogen for years.
  CAUTION: DMSO is a toxic product and should be handled with care and discarded.
7. Vials containing frozen PBMCs are thawed using a 37 °C water bath. Upon reaching the liquid phase, cells are immediately transferred into a 15 ml tube followed by slow addition 10 ml of cold (4 °C) RPMI-5% FBS while gently shaking the tube.
  CRITICAL: This transfer should happen immediately when the liquid phase is reached during thawing, as DMSO is harmful to the cells.
8. Spin down at 400 × g for 7 min at 4 °C. Afterwards, remove the supernatant and resuspend the pellet in 1 ml of cold (4 °C) RPMI 2% FBS.
9. Count the cells and plate 5 × 10⁵ cells per well in a 96-wells round bottom plate within a volume of 50 μl RPMI 2% FBS for phosphoflow.
  PAUSE POINT: Cells can be kept at 4 °C for 1-2 h before proceeding with the phosphoflow protocol at Step B1.
Collection of murine organs (Timing: 15 min per mouse)
1. Mice are sacrificed by cervical dislocation. The protocol allows for other sacrifice methods that do not affect the anatomical region of the organ of interest, including methods that involve anesthetics.
2. Peritoneal lavage. A midline incision over the abdomen is made to retract the skin, keeping the abdomen intact. A 10 ml syringe, containing 5 ml of ice-cold PBS and 5 ml of air, is attached to the 25-gauge × ⅝ inch needle. The peritoneum is subsequently washed by gently flushing and shaking upon full injection for 1 min. Afterwards, the peritoneal lavage is collected by aspirating the peritoneal cavity with the 10 ml syringe. The lavage is collected in a 15 ml tube and is kept on ice for further processing.
3. Spleen. The peritoneal cavity can be opened to carefully extract the spleen. The spleen is located at the left flank in the abdomen and can be extracted using tweezers.
4. Other organs. Next, the Peyer’s patches can be isolated from the small intestine using curved tip tweezers. Lastly, the femur is extracted by two cuts in the pelvic bone and dislocation of the knee. Harvesting of various other anatomical compartments, including bronchoalveolar lavage (BAL), lungs and lymph nodes as previously described (Li et al., 2017). All organs are placed in ice-cold PBS for further processing.
Preparation of single-cell suspension of various murine organs (Timing: 15 min per mouse)
1. Spleen tissue is mechanically disrupted using a 100 μm cell strainer on a 50 ml tube and the plunger of a 2 ml syringe. During and following processing of the spleen, strainers are flushed with 5 ml of RPMI 1640 Glutamax^TM supplemented with 2% (vol/vol) FBS (RMPI-2% FBS) to acquire all cells. Peyer’s patches are processed in a similar manner, using a 100 µm cell strainer and a Petri dish. Strainers are flushed with maximal 200 μl of RMPI-2% FBS to acquire all cells.
  CRITICAL: It is important to immediately put the tubes with single cell suspension back on ice or place them in the fridge at 4 °C after processing.
2. The femur is processed using a mortar and pestle, crushing the bone and acquiring the bone marrow cells in 3 ml of RMPI-2% FBS. The bone marrow cell suspension is transferred through a 100 μm cell strainer on a 50 ml tube to ensure a single cell solution without parts of bone. The bone marrow cell suspension is kept on ice for upcoming procedures.
3. Peritoneal lavage cells are centrifuged at 400 × g for 7 min at 4 °C and the supernatant is discarded. Peritoneal lavage cell pellets are resuspended in 1 ml of RMPI-2% FBS and are kept on ice for upcoming procedures.
4. A cell counting sample is taken from all organs to determine the exact number of cells in each sample.
  CRITICAL: To measure ex vivo basal levels of phosphorylation, cells are kept on ice without any stimulation. Immediately proceed to Step C1.
  PAUSE POINT: Cells can be kept at 4 °C for 1-2 h before proceeding with the phosphoflow protocol.
In vitro stimulation of single-cell suspension (Timing: 30 min-6 h)
1. Stimulation and staining of cells is performed in 96-wells round bottom plates, using 5 × 10⁵ per well (200,000-1,000,000 cells is technically most optimal). 5 × 10⁵ cells per well are transferred in a volume of 50 μl in each well on ice, keeping the cells all time at 4 °C.
2. For stimulation times of less than 1 h, the 96-wells round bottom plate is placed in a water bath at 37 °C. Make sure only the bottoms of the wells are in contact with the water. For stimulation times longer than 1 h, the stimulus is added while the cells are on ice and subsequently incubated at 37 °C and 5% CO₂. The optimal stimulation time for phosphotargets in human and murine samples are stated in the original paper (Rip et al., 2020) and Table 2.
  CRITICAL: Some phospho-targets in murine B cells, mainly downstream signaling molecules as NF-κB and AKT/S6, are receptive to temperature changes and mechanical stress due to isolation of single cells. Rest these samples at 37 °C, preferably in a CO₂ incubator, for 3 h prior to stimulation. This is critical for optimal detection and sensitivity of the downstream signaling read-outs. The pre-incubation for these targets on frozen PBMCs is not recommendable, as the viability of these cells is lower compared to fresh material.
  OPTIONAL: For experiments including in vitro treatment of samples with BCR signaling inhibitors, incubate cell suspensions 3 h at 37 °C with BCR signaling for effective pre-treatment. If the signaling target requires resting, pre-treatment can be performed during this resting time.
3. When stimulating cells in a water bath, wait 1-2 min to let the samples reach 37 °C. Add the stimulating agent by pipetting 50 μl of stimulus to the wells containing 50 μl of sample. The unstimulated control samples are supplemented with 50 μl RMPI-2% FBS as stimulation control. In addition, add 10 µl RPMI-2% FCS containing fixable live/dead viability stain 10 min prior to fixation.
  CRITICAL: As the fixable live/dead viability stain is light sensitive and is not optimal in RPMI medium, prepare this solution freshly and add to the cells within 15 min post preparation.
4. Dilute the eBioscience FoxP3 fixation and permeabilization buffer 1:1 with the supplied diluent. When the stimulation time is over, fix the cells by adding 100 μl of the diluted fixation and permeabilization buffer for 10 min at 37 °C.
  CAUTION: This product contains paraformaldehyde (PFA), a toxic and mutagenic product. It should be handled with care and discarded as hazardous waste.
  NOTE: The supplied fixation buffer is 4x concentrated and is diluted in the supplied diluent by combining both in equal volume. The diluted fixative solution is added in equal volume the cells to reach the recommended working concentration (50 µl of 4× fixation buffer + 50 µl of diluent to the 100 µl of cells in RPMI).
5. After 10 min of fixation, centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant.
6. Wash by adding 200 µl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards. Perform this step two times. Proceed to Step D1.
  CRITICAL: Two times washing is critical in order to remove all fixation and permeabilization buffer.
  PAUSE POINT: Proceed directly to staining for flow cytometry or keep the cells in a volume of 200 µl of eBioscience Wash Buffer at 4 °C. This storage of cells at 4 °C is possible up to 5 days, without impacting the quality of the samples.
Fixation of single-cell suspensions for ex vivo measurements (Timing: 30 min)
1. Parallel to stimulated cells, 5 × 10⁵ cells per well are transferred in a 96-wells round bottom plate at a volume of 100 µl per well on ice, keeping the cells at 4 °C at all times.
  CRITICAL: This procedure must always be performed at 4 °C, as this is a read-out of ex vivo activation and temperature alterations could be of great influence.
2. Samples are stained with the fixable live/dead viability stain for 10 min at 4 °C in the dark prior to fixation.
  CRITICAL: As the fixable live/dead viability stain is not optimal in RPMI medium, prepare this solution freshly and add to the cells within 15 min post preparation.
3. Fix cells with 100 µl of the diluted fixation and permeabilization buffer for 10 min at 4 °C.
4. After 10 min of fixation, centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant.
5. Wash by adding 200 µl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards. Perform this step two times.
  CRITICAL: Two times washing is critical in order to remove all fixation and permeabilization buffer.
  PAUSE POINT: Proceed directly to staining for flow cytometry or keep the cells in a volume of 200 μl of eBioscience Wash Buffer at 4 °C. This storage of cells at 4 °C is possible up to 5 days, without impacting the quality of the samples.
Flow cytometry procedures (Timing: 1-2 h)
1. After the last wash at Step B6 or Step C5, supernatant is removed and the staining mix can be added. Cells are incubated with 40 μl mix to intracellularly stain for cell surface markers such as CD3, B220/CD19, IgD and IgM in eBioscience Wash Buffer. An FcR-blocking agent is added to the staining mix to avoid non-specific binding of the antibodies. Cells are incubated in the dark for 30 min at 4 °C.
  CAUTION: Sodium azide is highly toxic and dangerous to the environment. It should be handled with care and discarded as hazardous waste.
  CRITICAL: Carefully resuspend the pellet in 40 μl mix, ensuring all cells are in suspension.
  NOTE: The staining can also be performed for 15 min at room temperature (RT, ~20 °C) in the dark.
2. Samples are washed by adding 200 μl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards.
3. Incubate the samples with 40 μl eBioscience Wash Buffer containing the phospho-target antibody of choice for 30 min at RT in the dark.
  OPTIONAL: If the phospho-target antibody is already labeled with a conjugate, proceed to Step D6 upon completion of the incubation time.
4. Samples are washed by adding 200 μl eBioscience Wash Buffer to all wells and centrifuge at 400 × g for 3 min at 4 °C. Carefully remove the supernatant afterwards.
5. When the phospho-protein concerns an unconjugated rabbit-derived antibody, incubate with a donkey anti-rabbit PE-conjugated antibody for 15 min at RT in the dark.
6. Samples are washed with MACS Buffer (0.5% BSA and 2 mM EDTA in PBS) and centrifuged at 400 × g for 3 min at 4 °C. Remove the supernatant and resuspended in 50 μl MACS buffer. Samples are transferred into insert tubes and measured using a flow cytometer within 1 day.
7. Prior to measuring, calibrate the LSR-II by compensating for all conjugates that are used within the phospho-staining. Preferably use the same antibodies as used in the staining mix and add those to UltraComp eBeads (Invitrogen) according to manufacturer instructions to calibrate the flow cytometer.
8. Acquire a sufficient number of cells to perform B cell subpopulation analysis. This would imply that acquiring between 20,000-50,000 B cells per sample would be sufficient.
  CRITICAL: Due to the fixation and permeabilization protocol, the cell size will have decreased compared with non-fixed cells. Increase the voltage of the forward-scatter area (FSC-A) accordingly.
9. Analysis is performed using FlowJo v9 or v10 software.

Data analysis

In this protocol manuscript, we include targets described in the original paper (Rip et al., 2020) and provide directions on the analyses of several additional phospho-targets that we recently established.

For each target, it is crucial to first find the best stimulus and identify the most optimal time of stimulation. Table 2 provides directions on the optimal stimulation time for BCR signaling molecules mentioned in the original paper (Rip et al., 2020) and additional phospho-targets in mice and men. During optimization of the subset and phospho-target staining procedure, all antibodies should be validated by including isotype and fluorescence minus one (FMO) controls, thereby verifying the signal intensities. Following acquisition of the cells by the flow cytometer, compensation can be checked and adjusted when necessary using the FlowJo 10 Software program.

As a data analysis example, Figure 1 provides an overview of the phosphorylation status analysis for various BCR signaling molecules in B cells from human PBMC fractions, either unstimulated or stimulated by anti-IgM F(ab')₂ fragments or recombinant CD40L. Prior to gating B cell subpopulations, we first selected for live, single lymphocytes followed by the identification of B cells with the pan-B cell marker CD19 (Figure 1A). We included CD3 in the staining to enable accurate gating of B cells as CD19⁺CD3^- cells. Moreover, CD19^-CD3⁺ T cell fractions can serve as a useful negative control for the analysis of phosphorylation of BCR signaling molecules as T cells are not responsive to anti-IgM stimulation. Within live CD19⁺ B cells, the populations of transitional B cells and plasmablasts were identified as CD27^-CD38⁺ and CD27⁺CD38⁺ cells, respectively. B cells with low CD38 expression were subsequently divided into naive CD27^-IgD⁺ B cells and CD27⁺IgD^- memory B cells (Figure 1A).

As a next step, phosphorylation of BCR signaling molecules was analyzed in gated B cell subpopulations. For example, we gated on CD27^-IgD⁺naive B cells and analyzed PLCγ2 (Y759) phosphorylation. For this analysis, next to FMO or isotype controls, gated T cells served as a negative staining control, because PLCγ2 is not expressed in T cells. The histogram overlays in Figure 1B show that the pPLCγ2 fluorescence signals of naive B cells were strongly increased upon anti-IgM stimulation, compared with unstimulated naive B cells. It is of note that unstimulated cells often show low but detectable ex vivo phosphorylation of various BCR signaling proteins. Phosphorylation signals were quantified by geometric mean fluorescence intensity (gMFI) values, because we observed stimulation-induced intensity shifts of a single peak in the case of pPLCγ2 (Figure 1B). In contrast, phosphorylation is quantified by calculating proportions of positive cells when a bimodal distribution is observed (for example for phosphorylation of ribosomal protein S6 (Figure 1C). We usually also include a vertical line for reference that is set either (i) at the peak of the counts of the unstimulated control, when stimulation results in an intensity shift of a single peak; or (ii) to indicate background signals, when stimulation increased the numbers of positive cells in a bimodal staining distribution (as in pS6).

Additional examples include the analysis in total CD19⁺ B cells fractions of phosphorylation of CD79a and ribosomal protein S6, a downstream target of the AKT signaling pathway (Figure 1C). Finally, we show histogram overlays of phosphorylation targets that were not previously described in Rip et al. (2020), including the activating Y418 (in mouse B cells: Y424) and inhibitory Y507 of Src-family kinases, PI3K p85 subunit (Y458) and the Src homology region 2 domain-containing phosphatase-1 (SHP-1; Y564) (Figure 1D).

Figure 1. Phosphorylation of BCR molecules in PBMC-derived B cells. A. Gating strategy for live single lymphocytes from the peripheral blood mononuclear cell (PBMC) fraction. Within lymphocytes, we gated for CD19⁺ cells (CD19⁺CD3^-). After exclusion of transitional B cells (Trans) and plasmablasts (PB), we gated for naive B cells (CD19⁺CD38^-IgD⁺CD27^-) and IgD-negative memory B cells (CD19⁺CD38^-IgD-CD27⁺). B. Histogram overlay for pPLCγ2 in naive B cells after 5 min of anti-IgM stimulation. C. Histogram overlay for pS6 after 30 min of anti-IgM or recombinant human CD40 ligand (CD40L) and IL-4 stimulation, and for pCD79a after 1 min of anti-IgM in total CD19⁺ B cells. D. Histogram overlays of total CD19⁺B cells for pSRC-Y418 after 1 min of anti-IgM stimulation and for pSRC-Y507, pPI3K p85 and pSHP-1 after 3 min of anti-IgM stimulation. Vertical lines in histograms are set on unstimulated controls as reference for stimulation, whereby the quantification by geometric mean fluorescence intensity (gMFI) is indicated. Only for ribosomal protein S6, which showed a bimodal distribution, phosphorylation is quantified by calculating proportions of positive cells (C). Graphs represent representative histograms of 2-10 individual experiments, each with 2-20 healthy donors per group.

In Figure 2 we present a parallel phospho-analyses on some previously validated (Rip et al., 2020) and novel targets for murine B cells. We selected live splenic B cells by gating on live B220⁺ cells and subsequently for naive B cells by IgD and IgM receptor expression (Figure 2A). Within naive B cells, we gated on follicular and marginal zone (MZ) B cells, using CD21 and CD23 (Figure 2A). Phosphorylation of PLCγ2 in follicular B cells showed about a 2.5-fold increase upon stimulation with anti-IgM (Figure 2B). Likewise, robust phosphorylation of CD79a following anti-IgM stimulation and of S6 following anti-IgM, anti-CD40 or lipopolysaccharide (LPS) stimulation was detected in naive B cells (Figure 2C). Additional BCR targets also displayed responsiveness following anti-IgM stimulation, with an optimal signal at 5 min for pSrc kinases (for both Y424 and Y507) and 1 min for pPI3K p85 and pSHP-1 (Figure 2D).

Figure 2. Phosphorylation of BCR molecules in splenic murine B cells. A.Gating strategy for live, single splenic lymphocytes. Within lymphocytes, we gated for splenic B220⁺ cells (B220⁺CD3^-). After purification for naive B cells using IgD and IgM, we gated for marginal zone (MZ) B cells (B220⁺CD21⁺CD23^-) and follicular (Fol) B cells (B220⁺CD21^-CD23⁺). B. Histogram overlay for pPLCγ2 after 5 min of anti-IgM stimulation in Fol B cells. C. Histogram overlays for pCD79 after 5 min of anti-IgM stimulation or pS6 after 3 h of anti-IgM, anti-CD40 or Lipopolysaccharide (LPS) stimulation. D. Histogram overlays of total naive B cells for pSrc-Y424 and pSrc-pY507 after 5 min and for pPI3K p85 and SHP-1 after 1 min of anti-IgM stimulation. Vertical lines in histograms are set on unstimulated controls as reference for stimulation, whereby the quantification by geometric mean fluorescence intensity (gMFI) is indicated. Only for ribosomal protein S6, which showed a bimodal distribution, phosphorylation is quantified by calculating proportions of positive cells (C). Graphs represent representative histograms of > 3 individual experiments, each with 5 mice per group; wild-type mice were 8-12 weeks old.

Notes

Timing for phosphorylation of other signaling proteins was presented in our original paper (Figures 1-3 and an schematic overview in Figure S2) (Rip et al., 2020).
It is not required to perform multiple washing steps, since we did not find differences in the quality of the staining between procedures with single or multiple washing steps. Single washing steps are therefore sufficient, unless otherwise indicated within the methods section.
The phosphoflow protocol was developed and validated for RPMI1640 medium. We did not investigate effects of other types of medium or supplements such as HEPES or 2-Mercapto-ethanol.
Dilutions and timing of the previously validated and additional targets shown in the Data analysis are included in Table 2. Note that both mAb dilution and timing for the phospho-antibody can vary between human and murine samples.
Phosphorylation of STAT protein and read-out for the NF-κB signaling pathway should be performed using other protocols, as mentioned in our original paper (Figures 2-3 and Figure S1) (Rip et al., 2020).
In downstream signaling pathways in freshly acquired murine B cells are receptive to temperature changes and mechanical stress due to isolation of single cells. Rest these samples at 37 °C prior to stimulation, preferably in an CO₂ incubator. We recommend to rest the samples 3 h, as is critical for optimal detection and sensitivity of the downstream signaling read-outs.
The pre-incubation for downstream signaling is not recommended on frozen PBMCs, as this strongly affects viability. We did not encounter this viability issue on frozen splenic suspensions derived from murine tissue.

Recipes

Solutions are not filtered; media, sera and buffers are sterile, fetal bovine serum (FBS) is heat-inactivated for 1 h at 56 °C prior to use.

Freezing medium
80% Fetal Bovine Serum
20% Dimethyl sulfoxide
RPMI-2% FCS
RMPI 1640
2% Fetal Bovine Serum
RPMI-5% FBS
RPMI 1640
5% Fetal Bovine Serum
MACS Buffer
Phosphate-buffered saline
0.5% Bovine Serum Albumin
2 mM Ethylenediaminetetraacetic acid solution

Acknowledgments

These studies were partially supported by the Dutch Cancer Society (KWF grant 2014-6564) and Target-to-B, both awarded to R.W. Hendriks, as well as an unrestricted grant from AcertaPharma B.V. Oss.

We would like to thank Allard Kaptein (AcertaPharma B.V., Oss), Stefan Neys and Marjolein de Bruijn (Erasmus MC Rotterdam) and the EDC Erasmus MC animal facility for their great technical assistance.

Author Contributions

JR designed the research, performed experiments, analyzed the data, and wrote the manuscript. OC and RH contributed to the research design and the writing of the manuscript and supervised the study. All co-authors approved the final manuscript.

Competing interests

The authors declare to have no financial conflicts of interest.

Ethics

PBMCs were obtained from healthy volunteers (Rotterdam Centrum, the Netherlands). Experimental procedures were approved by the ethics committee of the Erasmus MC.

Prior to animal experiments, all experimental protocols were reviewed and approved by the Erasmus MC Committee of animal experiments (DEC) and the central committee for animal experiments (CCD) with approval ID AVD1010020173764. CCD approval is valid until the 1^st of June 2023.

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