Bone marrow isolated from leukemic mice was blocked with anti-mouse Fc Block (BD Biosciences, dilution 1:50) for 10 min at 4 °C. Surface staining with anti-CD45-APC/Cy7 (dilution 1:100) and DPP4-PE (dilution 1:20) was performed at 4 °C for 30 min. Following this, the samples were washed in Medium-199 supplemented with 2% FBS and then fixed with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4 °C. The fixed cells were thereafter washed with 1× Perm/Wash buffer (BD Biosciences) and resuspended in Perm/Wash buffer containing anti-Ki67-AF647 at a 1:10 dilution for a 30 min incubation. The samples were washed one more time with 1× Perm/Wash buffer (BD Biosciences) and then incubated in 1× Perm/Wash buffer (BD Biosciences) with 2 μg ml−1 DAPI (Biolegend) for 10 min. Finally, the samples were spun down to remove the DAPI-containing buffer and resuspended in Medium-199 supplemented with 2% FBS for analysis. Flow cytometry was performed on a BD FACS Aria III sorter (BD Biosciences) and all data were analyzed using FlowJo (Treestar).
Perm wash buffer
Manufactured by BD
Sourced in United States, United Kingdom, Germany, Canada, France
Perm/Wash buffer is a laboratory reagent used in various biochemical and cell biology applications. It is designed to facilitate the permeabilization and washing of cells or cellular components. The buffer composition helps maintain the integrity and structure of the target samples during these processing steps.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm, by BD (170 mentions)
Cytofix cytoperm solution, by BD (130 mentions)
Cytofix cytoperm buffer, by BD (122 mentions)
Golgistop, by BD (63 mentions)
Ionomycin, by Merck Group (59 mentions)
Facscalibur, by BD (55 mentions)
Golgiplug, by BD (54 mentions)
Fixation permeabilization solution, by BD (48 mentions)
Facscanto 2, by BD (43 mentions)
Phorbol myristate acetate (pma), by Merck Group (42 mentions)
Cytofix cytoperm kit, by BD (41 mentions)
Cytofix fixation buffer, by BD (37 mentions)
Lsrfortessa, by BD (37 mentions)
Lsrii flow cytometer, by BD (36 mentions)
Brefeldin a, by Merck Group (33 mentions)
Facscalibur flow cytometer, by BD (31 mentions)
Lsr 2 flow cytometer, by BD (26 mentions)
Facscanto 2 flow cytometer, by BD (26 mentions)
Perm wash buffer, by Thermo Fisher Scientific (26 mentions)
Facsdiva software, by BD (25 mentions)
1 124 protocols using perm wash buffer
1
Immunophenotyping of Leukemic Mouse Cells
2
Mouse Liver Single-Cell Isolation and Immunostaining
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Mouse liver was perfused with 2% fetal bovine serum in phosphate buffered saline. Freshly harvested mouse liver tissue (about 0.5cm*0.5cm*0.3cm) was ground gently in PBS buffer containing 150 U/mlDNase I. Single-cell suspensions were generated by 100 μm cell strainer filtration. After centrifuge, cells were resuspended with 2% fetal bovine serum in PBS and was ready to use. Cells were then fixed and permeabilized by adding 500 μ;L of Fixation/Permeabilization solution (BD) and incubated at 20°C for 20 minutes. After washing the cells by adding 2 mL BD Perm/Wash buffer for two buffer exchanges, the cell pellet was resuspended in 100 μL BD Perm/Wash buffer containing goat anti human albumin primary antibody (Bethyl, A80-129A). Cells were immunostained with antibody for 30 minutes. After washing by adding 2 mL BD Perm/Wash buffer, the cell pellet was resuspended in 100 μL BD Perm/Wash buffer containing Allophycocyanin (APC) conjugated donkey anti goat IgG secondary antibody (R&D system). Cells were immunostained for 30 minutes at 20°C in the dark. After washing by PBS, cells were analyzed by flow cytometry.
3
Mouse Liver Single-Cell Isolation and Immunostaining
4
Multicolor Flow Cytometry Analysis of Stem Cell Markers
5
Apoptosis Induction by Ginger and PB
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After 24 h of treatment with the extracts of ginger and PB and their combination (ginger: 200, 500 and 800 µg/mL; PB: 200, 450 and 800 µg/mL; combination: 50, 100 and 200 µg/mL), the cells were harvested by trypsinization and then washed twice with cold 1X PBS.
The cells were then resuspended in BD Cytofix/Cytoperm solution and placed on ice for 20 min before a quick spin to isolate the pellet. The pellet was then washed twice with BD Perm/Wash buffer, resuspended in anticaspase-3 antibody and incubated for 30 min at room temperature. After washing with BD Perm/Wash buffer, the pellet was resuspended in BD Perm/Wash buffer and analysed by a Becton Dickinson FACSCalibur TM Flow Cytometer (Ontario, Canada).
The cells were then resuspended in BD Cytofix/Cytoperm solution and placed on ice for 20 min before a quick spin to isolate the pellet. The pellet was then washed twice with BD Perm/Wash buffer, resuspended in anticaspase-3 antibody and incubated for 30 min at room temperature. After washing with BD Perm/Wash buffer, the pellet was resuspended in BD Perm/Wash buffer and analysed by a Becton Dickinson FACSCalibur TM Flow Cytometer (Ontario, Canada).
6
Identification and Quantification of Quiescent LT-HSCs
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Bone marrow from femurs and tibias was depleted for red blood cells and cells expressing mature lineage markers as described above. Lineage-depleted BM was then stained for LT-HSCs using the following antibodies: Alexa-700 conjugated lineage antibodies (Ter119, B220, Mac1, Gr1, Ly6G and CD3), c-kit-APC, Sca1-PECy7, CD48-APCCy7 and CD150-PE, in FACS buffer (generated in house: PBS with 5% FCS (Hyclone) and 2mM EDTA) for 40’ on ice. Cells were washed with FACS buffer and then fixed in 500μL BD Cytofix/Cytoperm buffer (BD Biosciences) for 45 min on ice. Fixed cells were washed twice with 4mL 1x BD Perm/Wash buffer (BD Biosciences) by centrifugation for 5’ at 220 x g at 4°C and then stained with 20μL FITC-conjugated mouse anti-Ki67 antibody or isotype control (BD Biosciences) in the dark for 18 h at 4°C. Cells were washed twice with 1x BD Perm/Wash buffer (BD Biosciences), incubated with 10 μg/mL DAPI in 1x BD Perm/Wash buffer for 30’ on ice and then washed with and resuspended in FACS buffer for flow cytometry. Ki67 staining and DNA content was analyzed on the BD Symphony (BD Biosciences) and using FlowJo 10 software (TreeStar).
7
Caspase-3 Apoptosis Measurement in Breast Cancer Cells
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Caspase-3 was measured using the PE Active Caspase-3 Apoptosis Kit (San Diego, CA, USA)according to the manufacturer’s instructions. The MDA-MB-231 and ZR-75-1 cells were treated with 100 µg/mL of rGO for 24 and 48 h. Next, the cells were harvested and washed with cold 1× DPBS. Then, the cells were resuspended in Cytofix/Cytoperm solution at a concentration of 1 × 106 cells/0.5 mL. The cells were incubated for 20 min on ice. Pellet cells were aspirated, and the remaining cells were washed twice with Perm/Wash buffer (1×) at RT. The cells were resuspended in BD Perm/WashTM buffer (1×) plus antibody and incubated for 30 min. Washes were performed using 1 mL BD Perm/WashTM buffer (1×) before finally resuspending the cell solution in 0.5 mL BD Perm/WashTM buffer (1×). Data were acquired and evaluated with FACSDiva software.
8
Cell Surface and Intracellular Staining Protocol
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For surface staining, cells were washed twice with Dulbecco’s PBS (DPBS; Sigma-Aldrich), stained for 20 min on ice in 100 μl DPBS, washed twice in DPBS, and fixed for 20 min on ice in 100 µl Cytofix (BD Biosciences). For intracellular staining, cells were washed twice with DPBS, fixed, and permeabilized in 100 μl Cytofix/Cytoperm Solution (BD Biosciences) for 20 min on ice, washed twice in Perm/Wash buffer (BD Biosciences), stained for 20 min on ice in 100 μl Perm/Wash buffer, washed twice with Perm/Wash buffer, once with DPBS, and resuspended in DPBS. Cells were acquired using a Cyan ADP Analyser (Beckman Coulter) or an Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed using FlowJo 10.4. Details of antibodies used are listed in Table S2 .
9
Multi-Cytokine Profiling of Vaccine-Specific T-Cells
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Splenocytes were isolated 12 days after vaccination, plated at 1-2x106 cells/well in 96-well plates, and stimulated with vaccine proteins (HlaH35L, EsxAB, FhuD2 and Csa1A, 10 μg/ml each) together with anti-CD28 and anti-CD49d mAb (2 μg/ml each, BD Biosciences) at 37°C for 16–18 h. Brefeldin A (5 μg/ml) was added for the last 4 h. The cells were then stained with Live/DeadYellow (Invitrogen), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), washed in Perm/Wash buffer (BD Biosciences), incubated with anti-CD16/CD32 Fc block (BD Biosciences) for 20 min at RT, and stained with the following fluorochrome-conjugated mAbs anti: CD3-PerCP Cy5.5, CD4-V500, IFN-γ-PE, IL-2-APC, TNF-AF700, CD44-V450 (BD Pharmingen), CD8-PE Texas Red (Invitrogen), IL-17A-PE Cy7, IL-4-AF488 and IL-13-AF488 (eBioscience) in Perm/Wash buffer (BD Biosciences) for 20 min at RT, washed twice in Perm/Wash buffer, suspended in PBS. Samples were acquired on a LSRII special order flow cytometer (BD Biosciences) and T-cell responses were analyzed using FlowJo software (TreeStar) applying the gating strategy described in S1 Fig .
10
Cardiac Troponin T Quantification
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Cells were trypsinized and fixed with fixation buffer (BD Biosciences, San Jose, CA) for 15 minutes at room temperature. Fixed cells were washed with Perm/Wash buffer (BD Biosciences).8 , 9 Then, cells were incubated with primary mouse anti‐cTnT (cardiac troponin T) antibody (ab8295) in Perm/Wash buffer for 90 minutes at room temperature followed by incubation with donkey anti‐mouse Alexa Fluor 647 (ab150107). Cells were washed with Perm/Wash buffer, and then analyzed for cTnT expression using an LSR Fortessa cell sorter (BD Biosciences, Franklin Lakes, NJ) with FlowJo software (Flowjo, LLC, Ashland, Ore).
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