Cytofix cytoperm plus kit with golgiplug

Manufactured by BD

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The BD Cytofix/Cytoperm Plus Kit (with BD GolgiPlug) is a laboratory reagent used for the intracellular staining and flow cytometric analysis of proteins. The kit includes a fixation and permeabilization solution, as well as a protein transport inhibitor.

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7 protocols using cytofix cytoperm plus kit with golgiplug

Multiparametric Flow Cytometry of Tumor-Infiltrating Immune Cells

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For the analysis of immune cells from ex vivo tumor cultures, antibodies specific for human CD3 (clone SK7, Alexa Fluor 700, Biolegend), CD4 (clone RPA-T4, V500, BD Biosciences, NJ, USA), CD8 (clone RPA-T8, FITC, BD Biosciences, Franklin Lake, NJ, USA), CD69 (clone FN50, PE/Cyanine7, Biolegend), granzyme B (GrzmB) (clone GB11, BV421, BD Biosciences, NJ, USA), perforin (Perf) (clone B-D48, PerCP/Cyanine5.5, Biolegend, San Diego, CA, USA)), and CD107a (LAMP1) (clone H4A3, APC/Cyanine7, Biolegend, NJ, USA) were used. Intracellular staining was performed using BD Cytofix/Cytoperm Plus Kit (with BD GolgiPlug) (BD Biosciences). All samples were stained after Fc blocking using Human TruStain FcX Receptor Blocking Solution (Biolegend, San Diego, CA, USA). Samples were acquired in duplicates using FACS Aria II cell sorter (BD Biosciences, Franklin Lake, NJ, USA) and data analysis was performed using Flowjo software v10 (Flowjo LLC, BD Biosciences, Franklin Lake, NJ, USA).

Heiniö C., Clubb J., Kudling T., Quixabeira D., Cervera-Carrascon V., Havunen R., Grönberg-Vähä-Koskela S., Santos J.M., Tapper J., Kanerva A, & Hemminki A. (2022). Effective Combination Immunotherapy with Oncolytic Adenovirus and Anti-PD-1 for Treatment of Human and Murine Ovarian Cancers. Diseases, 10(3), 52.

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Multiparametric Analysis of Tumor-Infiltrating Immune Cells

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Analysis of immune cells from hamster’s tumor single-cell suspensions was performed using the following cross-reactive antibodies: PE-Cyanine7 anti-mouse CD4, PE anti-rat CD8b, Alexa Fluor 488 polyclonal anti-asialo-GM1, FITC anti-mouse/rat MHC class II (I-Ek) (Supplementary table S2).
Analysis of immune cell populations from ex vivo tumor cultures was performed using the following antibodies: V500 anti-human CD4, FITC anti-human CD8, PE-Cy7 anti-human CD69, PE-CF594 anti-human CD127, BV421 anti-human Granzyme B, PerCP-Cyanine5.5 anti-human Perforine, APC-Cyanine7 anti-human CD107a antibodies (Supplementary table S2). Intracellular staining was performed using BD Cytofix/Cytoperm Plus Kit (with BD GolgiPlug) (BD Biosciences, New Jersey, USA), according to manufacturer’s instructions. Samples were stained after Fc blocking using Human TruStain FcX Receptor Blocking Solution (Biolegend, California, USA).
Analysis of immune cells from murine PDX-OvCa tumors was performed using the following antibodies: Alexa Fluor 700 anti-human CD3, V500 anti-human CD4, FITC anti-human CD8, PE-Cy7 anti-human CD69 (Supplementary table S2).
All samples were acquired in duplicates using FACS Aria II cell sorter (BD Biosciences, New Jersey, USA) collecting at least 50000 events per sample. Data analysis was performed using Flowjo software v10 (Flowjo LLC, BD Biosciences, New Jersey, USA).

Kudling T.V., Clubb J.H., Quixabeira D.C., Santos J.M., Havunen R., Kononov A., Heiniö C., Cervera-Carrascon V., Pakola S., Basnet S., Grönberg-Vähä-Koskela S., Arias V., Gladwyn-Ng I., Aro K., Bäck L., Räsänen J., Ilonen I., Borenius K., Räsänen M., Hemminki O., Rannikko A., Kanerva A., Tapper J, & Hemminki A. (2022). Local delivery of interleukin 7 with an oncolytic adenovirus activates tumor-infiltrating lymphocytes and causes tumor regression. Oncoimmunology, 11(1), 2096572.

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Single-cell immune profiling by flow cytometry

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Single cell suspensions were stained and analyzed on a BD LSR II cytometer. Antibodies were purchased from BD bioscience unless otherwise listed. For monocyte, neutrophil, DC, macrophage and T cell stainings, the following antibodies were used: anti-Ly6C (clone AL-21), Ly6G (1A8), CD11b (M1/70), CD45 (30F-11), CD11c (HL3), CD103 (2E7), NK1.1(PK136), CD49b(PanNK)(DX5), CD3 (145-2C11), CD4 (L3T4), γδ TCR (GL3), TNF (MP6-XT22), IL-17RA (PAJ-17R). For ILC staining, the following antibodies were used: Lineage 1(Lin1, in FITC): CD3, CD4, CD8 (53–6.7), CD11c; Lineage2 (Lin2, in PE-CY7): CD11b, NK1.1, CD19 (1D3), Ter119 (TER119). CD45, CD45.1 (A20), CD45.2 (104), CD90 (53–2.1), CD127 (A7R34) were used for the subsequent positive gating.
For intracellular TNF staining, BD Cytofix/Cytoperm Plus Kit (with BD GolgiPlug) (cat# 555028) with Protein Transport Inhibitor (Containing Brefeldin A) (cat#555029) was used. Brefeldin A was added in the digestion and staining buffers. For intracellular IL-17A and IL-22 staining, the antibodies were purchased from ebioscience (Clone: eBio17B7, cat# 17–7177-81 for anti IL-17 and Clone: IL22JOP cat# 17–7222-82 for anti IL-22). Intracellular Fixation & Permeabilization Buffer (plus Brefeldin A) (cat# 88–8823-88) was used. Cells were stimulated in vitro with ionomycin (750ng/ml), PMA (50ng/ml) and IL-23 (40ng/ml) for 3 hours before IL-17A/IL-22 staining.

Xiong H., Keith J.W., Samilo D.W., Carter R.A., Leiner I.M, & Pamer E.G. (2016). Innate lymphocyte/Ly6Chi monocyte crosstalk promotes Klebsiella pneumoniae clearance. Cell, 165(3), 679-689.

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Flow Cytometric Analysis of IL-4+ CD4+ T Cells

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To assess the recruitment of IL-4⁺ CD4⁺ T cells, live cells were isolated from splenocytes and LLN from allergic airway inflammatory mice that were or were not sensitized to the A. pegreffii crude extract. The cell preparation method was the same as that described in Section 2.8. Samples were measured and analyzed on a flow cytometer (3-laser, 10-color; SONY SA3800) using the appropriate mAbs. The antibodies used for cell surface staining included purified rat anti-mouse CD16/CD32 (553142; Mouse BD Fc Block™, BD Pharmingen™), CD4 monoclonal antibody (T helper cell marker, 17-0042-82; RM4-5, APC, eBioscience, San Diego, CA, USA), and rat IgG2a kappa isotype control (17-4321-81; eBR2a, APC, eBioscience), while intracellular staining was performed with PE-Cy™7 rat anti-mouse IL-4 (560699; BD Pharmingen™), PE-Cy™7 rat IgG1κ isotype control (557645; BD Pharmingen™), anti-iNOS-PE cyanine7 (25-5920-82), and anti-arginase 1-PerCP-eFluor 710 (46-3697-82; eBioscience). Additionally, the Intracellular Fixation and Permeabilization Buffer (BD Cytofix/Cytoperm Plus Kit with BD GolgiPlug, 555028; BD Pharmingen™) was used. The experiment was set up according to the recommendations of BD Pharmingen. During sample gating, cells were gated against LLN. The LLN gate determined CD4⁺ cells. IL-4⁺ T cell expression was determined from the gated population.

Choi J.H., Kim J.Y., Yi M.H., Kim M, & Yong T.S. (2021). Anisakis pegreffii Extract Induces Airway Inflammation with Airway Remodeling in a Murine Model System. BioMed Research International, 2021, 2522305.

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Multiparameter Flow Cytometry Analysis

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Cells were first stained with fluorochrome-conjugated monoclonal antibodies (mAb) against cell surface markers. Fc receptors were blocked using supernatant from anti-FcgIIIR mAb-secreting 2.4G2 cell line and intracellular IFN-γ or IL-2 detected using a Cytofix/Cytoperm Plus kit with GolgiPlug (BD) according to the manufacturer's instructions. In assays detecting CD107a, this antibody was added to the cells/medium at the start of stimulation. Stained cells were acquired on a BD LSRII flow cytometer and analyzed with FACSDIVA software. The antibodies used were anti-IFN-γ-APC (BD), anti-IL2-PE (BD), anti-TCRβ-APC-eFluor-780 (eBiosciences, Hatfield, UK), anti-CD4-PerCP-Cy5.5 (BD), anti-CD107a-PE (BD), and anti-CD8-FITC (BD). Expression of MHC class I molecules on tumor cells was quantified by staining with FITC-conjugated anti-H2-D^d (BD Pharmingen) and PE-conjugated anti-H-2K^d (BD Pharmingen, Oxford, UK). To quantify LMP2A expression by tumor cells, the cells were first fixed with 4% v/v paraformaldehyde, permeabilized with 0.1% wt/v saponin solution in PBS and stained with 1:100 dilution of a rat monoclonal antibody to LMP2A (14B7), followed by a 1:100 dilution of PE-conjugated goat anti-rat IgG (Invitrogen). Cells stained with secondary antibody alone were used as controls.

Ondondo B., Faulkner L., Williams N.A., Morgan A.J, & Morgan D.J. (2015). The B subunit of Escherichia coli enterotoxin helps control the in vivo growth of solid tumors expressing the Epstein–Barr virus latent membrane protein 2A. Cancer Medicine, 4(3), 457-471.

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Characterizing Lung Immune Cells

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BAL was collected after injecting 1 mL of PBS. Total and differential cell counts were determined by hemocytometer and cytospin preparation and stained with Instant-Prov (Newprov, Brazil). Lung tissue was digested using collagenase IV (2 mg/mL) and DNase I (1 mg/mL) (Sigma-Aldrich) at 37 °C for 30 min. Single cell suspension was obtained after erythrocyte depletion, smashing and filtering. Total lung cells were restimulated ex vivo with 100 ng/mL PMA and 750 ng/mL ionomicin (Sigma) for 4 h or 1 μg/mL subtilisin for 18 h both in RPMI-1640 medium at 37 °C.
Single-cell suspensions from BAL and lung were blocked with 2.4G2 and incubated for 25 min at 4 °C with antibodies. A Cytofix/Cytoperm Plus kit with GolgiPlug (BD Pharmingen) was used for intracellular cytokine staining according to the manufacturer's instructions. Cell acquisition was performed on Canto II or LSRII instruments (BD) and data were analyzed with FlowJo software (TreeStar).

Florsheim E., Yu S., Bragatto I., Faustino L., Gomes E., Ramos R.N., Barbuto J.A., Medzhitov R, & Russo M. (2015). Integrated Innate Mechanisms Involved in Airway Allergic Inflammation to the Serine Protease Subtilisin. Journal of immunology (Baltimore, Md. : 1950), 194(10), 4621-4630.

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Cytokine Profiling of Monocytes

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Cell surface and intracellular cytokine staining of monocytes was performed as previously described (46 (link)). Briefly, cultured monocytes or purified monocytes were washed in PBS and stained by PE-conjugated anti-CD80 (BD Pharmingen, 557227), FITC-conjugated anti-CD86 (BD Pharmingen, 555657), PE-conjugated anti-CD163 (BD Pharmingen, 556018), FITC-conjugated CD206 (BD Pharmingen, 551135), PE-Cy7–conjugated HLA-DR (BD Pharmingen, 560651), or APC-conjugated anti-CD42b (BD Pharmingen, 551061) before analysis by FACSCanto. Fixation and permeabilization were performed using Cytofix/Cytoperm Plus kit with GolgiPlug (BD Biosciences) and stained by PE-conjugated anti–IL-1β (R&D Systems, IC201P) and PE-CF594–conjugated anti–IL-10 (BD Horizon, 562400) before analysis. All data acquired from flow cytometry were analyzed with FlowJo (Treestar software).

Li T., Yang Y., Li Y., Wang Z., Ma F., Luo R., Xu X., Zhou G., Wang J., Niu J., Lv G., Crispe I.N, & Tu Z. (2022). Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein. The Journal of Clinical Investigation, 132(4), e150101.

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