Murine aortic cells were retrieved through enzymatic digestion with collagenase I, collagenase XI, hyaluronidase, DNAse I and HEPES solution (Sigma-Aldrich, St. Louis, MO, USA) in a thermocycler for 45 min at 750 rpm and 37 °C. Murine blood samples were lysed in RBC lysis buffer (Biolegend, San Diego, CA, USA). Isolated cells from the blood and aorta were counted using a Neubauer chamber (Marienfeld, Lauda-Königshofen, Germany). Cells were stained with specific fluorescent antibodies as indicated (Supplemental Table 3). Ly6Chigh monocytes were identified as CD45+ CD11b+, Lin− (Lin = CD3, CD19, NK1.1, Ly6G), Ly6Chigh, CD115+, F4/80low. Macrophages were identified as CD45+ CD11b+, Lin−, Ly6Clow, F4/80high. Intracellular staining with anti-Ki67 and anti-active Caspase 3, BD Cytoxfix/Cytoperm (#554,722, BD Biosciences, San Diego, CA, USA), BD Perm/Wash (#554,723) and BD Permeabilization Buffer Plus (#561,651) was conducted according to the manufacturer’s instructions. Data were collected on a BD Facs Canto II (BD Bioscience, San Diego, CA, USA) and analyzed with FlowJo (Treestar, Ashland, OR, USA).
Permeabilization buffer plus
Manufactured by BD
Sourced in United States
Permeabilization Buffer Plus is a laboratory reagent designed to permeabilize cell membranes. It facilitates the entry of molecules, such as antibodies or dyes, into the intracellular space for various applications, including flow cytometry and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Fixation permeabilization solution kit, by BD (3 mentions)
Facscalibur, by BD (2 mentions)
Perm wash, by BD (1 mentions)
Hepes solution, by Merck Group (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Neubauer chamber, by Marienfeld (1 mentions)
Software, by FlowJo (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm plus fixation permeabilization kit, by BD (1 mentions)
Stemmacs media, by Miltenyi Biotec (1 mentions)
P smad2 s465 467 antibody, by Cell Signaling Technology (1 mentions)
Apotome 2, by Zeiss (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Annexin 5 binding buffer 1x, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
6 protocols using permeabilization buffer plus
1
Murine Immune Cell Isolation and Flow Cytometry
2
Ppp4r2 Knockdown Impacts DNA Damage
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Cells with either Ppp4r2 knockdown/PPP4R2 re-expression or control were exposed to 2 Gy IR or not followed by fixation and permeabilization at indicated time post IR with BD Cytofix/Cytoperm Buffer and BD Permeabilization Buffer Plus, respectively (Fixation/Permeabilization Solution Kit, BD Biosciences, Franklin Lakes, NJ, USA).
For measurement of pKAP1, cells were incubated with anti-pKAP1 antibody (S824; Bethyl Laboratories, Montgomery, TX, USA) and subsequently stained with anti-Dylight 649 donkey anti-rabbit IgG (BioLegend, San Diego, CA, USA).
Determination of γH2AX was performed by incubation with anti-phospho-Histone H2A.X antibody (Ser139; Clone JBW 301; Merck Millipore, Darmstadt, Germany) followed by staining with anti-Mouse IgG1 eFluor® 660 (eBioscience, San Diego, CA, USA).
Cells were acquired by flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by Flow Jo Software (FlowJo, Ashland, OR, USA).
For measurement of pKAP1, cells were incubated with anti-pKAP1 antibody (S824; Bethyl Laboratories, Montgomery, TX, USA) and subsequently stained with anti-Dylight 649 donkey anti-rabbit IgG (BioLegend, San Diego, CA, USA).
Determination of γH2AX was performed by incubation with anti-phospho-Histone H2A.X antibody (Ser139; Clone JBW 301; Merck Millipore, Darmstadt, Germany) followed by staining with anti-Mouse IgG1 eFluor® 660 (eBioscience, San Diego, CA, USA).
Cells were acquired by flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by Flow Jo Software (FlowJo, Ashland, OR, USA).
3
Quantifying Sca1+ Cell Uptake of PKH67+ EVs
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Four hours following the incubation of Sca1+ cells with PKH67+ EVs (109 particles per 4 × 105 cells) in the StemMACS media (Miltenyi Biotec), cells were stained with cell surface markers (SLAM). Cells were fixed and permeabilized with BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and Permeabilization Buffer Plus (BD Biosciences). We used the p-Smad2 (S465/467) antibody (#18338, Cell Signaling Technology) and the secondary anti-Rabbit-AF568 antibody (Thermo Fisher Scientific). Then, SLAM cells were purified by FACS and applied on a glass slide (Superfrost plus, Thermo Fisher Scientific) for up to 10 min and fixed with ProLong Gold Antifade reagent containing DAPI (P36931, Thermo Fisher Scientific). Fluorescent images were acquired with an Axio Imager M2 (Zeiss). Fluorescent optical sections of cells were obtained under magnification ×63 using an Axio Imager M2 (Zeiss) coupled with an Apotome.2 (Zeiss). Fluorescent images were processed for study (Fiji, NIH software).
4
Apoptosis and Cell Cycle Analysis of BM LSK Cells
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BM LSK cells (setting 2) were stained with Annexin-V and DAPI in Annexin V binding buffer 1X (BD Biosciences) for apoptosis analysis. BM LSK cell cycle was assessed by flow cytometry using Ki-67 and DAPI staining, after fixation and permeabilization (BD Cytofix/Cytoperm and Permeabilization Buffer Plus, BD Biosciences).
5
Multiparameter Apoptosis Profiling
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Thawed cells were stained with Zombie NIR Fixable Viability stain in PBS (BioLegend, cat #423105), followed by staining with cell-surface antibodies (see Supplementary Table S6). Stained cells were fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences, cat #554714) according to the manufacturer's instructions, followed by a secondary permeabilization step for enhanced intracellular staining using Permeabilization Buffer Plus (BD Biosciences, cat #561651). Fixed and permeabilized cells were stained separately for anti-human-BCL2-PE (clone 124, Cell Signaling Technology, cat #26295S), anti-human-MCL1-PE (clone D2W9E, Cell Signaling Technology, cat #65617S), and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S), or together for anti-human-BCL2-AF647 (clone 124, Cell Signaling Technology, cat #82655), anti-human-MCL1-AF488 (clone D2W9E, Cell Signaling Technology, cat #58326) and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S). Samples were analyzed with BD LSRFortessa Cell Analyzer.
6
Flow Cytometric Analysis of Immune Cells
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For flow cytometric analysis, the following monoclonal antibodies (mAbs) and reagents were used: CD8a (53–6.7), CD11b (M1/70), CD44 (IM7), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), B220 (RA3-6B2), Thy1.2 (30-H12), Gr-1 (RB6-8C5), rat-CD2 (OX34), Hamster IgG (HTK888), Notch1 (HMN1-12), Notch2 (HMN2-35), Notch3 (HMN3-133), Notch4 (HMN4-14), anti-Rabbit-IgG (Poly4064), mIgG1 (MOPC-21), AnnexinV and 7-AAD were purchased from BioLegend. CD25 (PC61.5), CD45 (30-F11), TER119 (TER-119), Gr-1 (RB6-8C5), CD11b (M1/70), CD11c (N418), CD19 (1D3), NK1.1 (PK136), CD3e (145–2 C11), hNGFR (ME20.4) were purchased from eBioscience. CD4 (GK1.5, RM4-5) and GATA3 (L50-823) were purchased from BD Biosciences. CD19 (1D3) was purchased from Tonbo Biosciences. TCF1 (C63D9), c-Myc (D84C12) and Rabbit-IgG (DA1E) were purchased from Cell Signaling Technology. LMO2 (EP3257) was purchased from Abcam. For intracellular staining, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Set (eBioscience) or Fixation/Permeabilization Solution Kit with BD GolgiStop (BD Biosciences) and Permeabilization Buffer Plus (BD Biosciences). Stained cells were measured with FACSCalibur (BD Biosciences) or FACSVerse (BD Biosciences). Data were analyzed using FlowJo (BD Biosciences).
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