Peripheral blood was collected at baseline (pre-treatment), C2D1, and post-cycle 3 (C4D1 or C6D1) for flow cytometry analysis. Peripheral blood was obtained using heparin collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated within 6 h using Ficoll-Paque Separation according to manufacturer’s instructions (GE Healthcare). PBMCs were cryopreserved in 10% DMSO, 90% FBS and thawed rapidly for flow cytometry analysis. Single-cell suspensions were prepared on ice in 2% FBS in PBS. Antibody cocktails were diluted in Brilliant Violet Buffer (BD Biosciences). Samples were acquired using a Cytek Aurora spectral cytometer with user settings established by Spectroflo QC beads. Unmixing and compensation were performed in Spectroflo software using a mix of reference controls from either single stained PBMCs or single stained OneComp compensation beads (eBioscience). Samples were stained with fluorescently tagged antibodies (Supplemental Table 4). Antibodies were titrated for optimal signal-to-noise ratio prior to use. Flow cytometry analysis was performed using Flowjo vX, and the CATALYST R package was used for FlowSOM analysis and UMAP projections [21 (link)]. All samples were gated on live, single cells.
Ficoll paque separation
Manufactured by GE Healthcare
Sourced in Sweden
Ficoll-Paque is a density gradient medium used for the separation and isolation of cells, such as mononuclear cells, from whole blood or other biological samples. It facilitates the separation of different cell types based on their density during centrifugation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
Liberase, by Roche (2 mentions)
M csf, by Thermo Fisher Scientific (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Dnase, by Merck Group (2 mentions)
Brilliant violet buffer, by BD (1 mentions)
Onecomp compensation beads, by Thermo Fisher Scientific (1 mentions)
Daudi, by American Type Culture Collection (1 mentions)
Karpas422, by American Type Culture Collection (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Mil 3, by Thermo Fisher Scientific (1 mentions)
Mil 6, by Thermo Fisher Scientific (1 mentions)
Foxp3 permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Tcs sp5, by Leica (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
15 protocols using ficoll paque separation
1
Multiparametric Flow Cytometry Analysis
2
Isolation and Culture of PBMCs, Lymphoma, and Lung Cancer Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Uppsala University Hospital, Uppsala, Sweden) by Ficoll‐Paque separation (GE Healthcare Life Sciences, Uppsala, Sweden) and cultured in RPMI‐1640 medium supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 2 mM l ‐glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 IU penicillin/ml & 100 μg streptomycin/ml (1% PeSt), and 20 μM β‐mercaptoethanol. The CD19‐positive human B‐cell Burkitt's lymphoma cell line Daudi, the CD19‐positive human B‐cell non‐Hodgkin's lymphoma cell line Karpas 422, and the human lung carcinoma cell line A549 were purchased from ATCC (Manassas, VA) and cultured in RPMI‐1640 supplemented with 10% heat‐inactivated FBS (20% for Karpas 422), 1 mM sodium pyruvate, and 1% PeSt. The human embryonic kidney cell line 293T (ATCC) was cultured in DMEM supplemented with 10% heat‐inactivated FBS, 1 mM sodium pyruvate, and 1% PeSt. All components and culture media were purchased from Invitrogen (Carlsbad, CA). All cells were cultured in humidified incubator with 5% CO2 at 37°C and regularly screened for mycoplasma infection (MycoAlert™ Mycoplasma Detection Kit from Lonza).
3
Mononuclear Cell Isolation Protocol
4
Fluorescent Mouse Bone Marrow Cells
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Bone marrow cells were isolated from femurs and tibias of C57BL/6J adult mice by flushing bone marrow with PBS. Red blood cells were removed through Ficoll-Paque separation (GE Healthcare). Cells were then washed and resuspended in complete media supplemented with 100 ng of mouse stem cell factor (m-SCF), 20 ng of m-IL-3 and 20 ng of m- IL-6 (Peprotech) and incubated over night at 37 °C. These cells were retrovirally transduced to express either the fluorescence resonance energy transfer (FRET)-based Twitch2B calcium indicator (17 (link)) or calcium actuator mScarlet-OptoSTIM-1 (eOS1) (18 (link)). Wild-type C57BL/6J recipient mice were γ-irradiated with a single dose of 9 Gy. Four hours later, irradiated mice were reconstituted with a total of 4 × 106 transduced bone marrow cells via intravenous (i.v.) injection. Mice were used 6 to 12 wk after reconstitution. Reconstitution was confirmed by flow cytometry by staining blood cells with a Alexa647-conjugated anti-Ly6G-mAb (clone 1A8, BD).
5
Neutrophil Isolation and Activation Assay
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Enriched human neutrophil fractions were isolated from heparinized blood from two healthy donors, as previously described (45 (link)). Briefly, after Ficoll-Paque separation (GE Healthcare), granulocytes were twice treated with red blood cell lysis using ACK lysing buffer (Thermo Fisher Scientific). Primary human neutrophils (0.2 × 106) were cultured in Hanks' balanced salt solution without calcium/magnesium for 4 h on 6 mm coverslips coated with 0.02% poly-L-Lysine (Sigma-Aldrich). Neutrophils were left untreated (medium) or stimulated with ionomycin (1 μM, Calbiochem) or phorbol 12-myristate 13-acetate (PMA) (50 nM, Calbiochem), then fixed with 3.7% formaldehyde and incubated in PBS-glycine (10 mM). Cells were permeabilized (eBioscience, Foxp3 permeabilization buffer), stained with murine chimeric ACPA IgG2a or control mAbs (10 μg/ml), and subsequently with anti-mouse-AlexaFluor488 (Invitrogen) and Hoechst (Thermo Fisher Scientific). Coverslips were mounted onto glass slides using Fluoromount-G (Southern Biotech). Images were acquired using a Leica TCS SP5 and a 63x oil objective. A z-dimension series was taken every 0.2 μm. Images were analyzed with the Fiji software.
6
Immune Cell Isolation from Blood and Tissue
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Patient peripheral blood was obtained by venipuncture using heparin collection tubes, transported at room temperature from the clinic to the lab, and processed within 6 hours of drawing. PBMCs were isolated via Ficoll-Paque separation (GE Healthcare, now Cytiva) following the manufacturer’s instructions. Solid tissue specimens were collected by surgical resection and collected in tubes containing cold HBSS (Life Technologies, Thermo Fisher Scientific) and transported on ice to the laboratory for processing within 1 hour of surgery. T– LNs were mechanically dissociated and filtered into single-cell suspensions. Tumor, T+ LNs, and NCBTs were minced into pieces; mechanically dissociated with a gentleMACS Dissociator (Miltenyi Biotec); and enzymatically treated with 0.2 Wunsch U/mL Liberase TM (Roche) and 10 U/mL DNase (MilliporeSigma) in RPMI for up to 1 hour as needed. If necessary, RBC lysis was performed using RBC Lysis Buffer (BioLegend).
7
Monocyte-Derived Macrophage Differentiation and Protein Overexpression
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The whole blood was collected from the healthy donors, and then the monocytes were isolated by Ficoll-Paque separation (GE Healthcare). The collected monocytes were differentiated in the presence of 100 ng/ml M-CSF (PeproTech, London, U.K.) for 3 days. Non-adherent cells were washed off, and the remaining adherent cells (monocytes derived macrophages) were maintained in RPMI 1640 medium supplemented with 20% heat-inactivated fetal calf serum, 1% penicillin/streptomycin, and 2% l-glutamine. To overexpress recombinant human DC-SIGN (with FLAG) and TLR4 (with His) proteins, HEK293 cells (ATCC, U.S.A.) were seeded onto 60-mm dishes at a density of 5.0 × 105 cells and cultured in DMEM medium containing 10% FBS.
8
Isolation of Wound Macrophages and Human Monocytes
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MACS of wound cell isolates was performed as described (70 (link)). Briefly, wound cell isolates were incubated with fluorescein isothiocyanate–labeled (FITC-labeled) anti-mouse anti-CD3, anti-NK1.1, anti-CD19, and anti-Ly6G (BioLegend) monoclonal antibodies. Wound isolates were then washed and incubated with anti-FITC microbeads (Miltenyi Biotec, Inc, catalog 130-049-601) and passed through a MACS column (Miltenyi Biotec, Inc). The resultant eluent was then incubated with anti-mouse anti-CD11b microbeads (Miltenyi Biotec, Inc, catalog 130-049-601). The remaining cell population was analyzed by flow cytometry and found to be 97% macrophages, consistent with previous literature (4 (link), 70 (link)). For human monocyte isolation, peripheral blood was collected and subjected to RBC lysis and Ficoll-Paque separation (GE Healthcare). Cell suspensions were then treated with anti-human CD14 microbeads. Magnetic separation yielded 95% purity by flow cytometry.
9
Isolation of PBMCs and Tumor Cells
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Patient peripheral blood was obtained by venipuncture using heparin collection tubes, transported at room temperature from the clinic to the lab, and processed within 6 h of drawing. PBMCs were isolated via Ficoll-Paque Separation (GE Healthcare) following the manufacturer’s instructions.
Tumor specimens were collected by surgical resection and collected in tubes containing cold RPMI (Life Technologies, Thermo Fisher Scientific) and transported on ice to the laboratory for processing within 1 h of surgery. Tumor tissues were minced into pieces and further mechanically dissociated with a gentleMACS Dissociator (Miltenyi Biotec). Tissue was treated with 0.2 Wunsch U/ml Liberase TM (Roche) and 10 units/ml DNase (Sigma) in RPMI for up to 1 h as needed. If necessary, red blood cell (RBC) lysis was performed using RBC Lysis Buffer (Biolegend).
Tumor specimens were collected by surgical resection and collected in tubes containing cold RPMI (Life Technologies, Thermo Fisher Scientific) and transported on ice to the laboratory for processing within 1 h of surgery. Tumor tissues were minced into pieces and further mechanically dissociated with a gentleMACS Dissociator (Miltenyi Biotec). Tissue was treated with 0.2 Wunsch U/ml Liberase TM (Roche) and 10 units/ml DNase (Sigma) in RPMI for up to 1 h as needed. If necessary, red blood cell (RBC) lysis was performed using RBC Lysis Buffer (Biolegend).
10
Bone Marrow Aspirates for AL Amyloidosis
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Bone marrow aspirates were collected from patients with treatment-naive and relapsed AL amyloidosis under protocol H-36533 at Boston Medical Center, which was approved by Panel Green Institutional Review Board (IRB) initially on 07/06/2017 and renewed annually for the duration of the studies. All patients provided statements of informed consent prior to sample collection. Mononuclear cells were isolated via Ficoll–Paque separation according to manufacturer’s instructions (GE Healthcare) and used for downstream applications.
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