Peripheral blood from healthy individuals was collected in lithium heparin coated Vacuette tubes (Greiner Bio-One, Kremsmuenster, Austria) or was provided as buffy coats from the Australian Red Cross Blood Service. For most experiments, MNCs were isolated using Lymphoprep. However, for analysis of VEGFR2+ EPCs, whole blood was subjected to erythrocyte lysis using PharmLyse (BD, Franklin Lakes, NJ, USA) followed by depletion of mature leucocytes using the Lineage Cell Depletion kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Primary HUVEC were extracted from human umbilical veins by collagenase digestion and cultured in HUVE medium as previously described [23 (link), 24 (link)] and were used for no more than two passages. Human bone marrow endothelial cells (TrHBMEC) were a kind gift from B Weksler (Cornell University Medical College, NY, USA) [25 (link), 26 (link)] and hereafter labelled as BMEC. Normal human bone marrow samples were pre-filtered through a 70-µm nylon filter (BD Falcon) to remove debris and then subject to red blood cell lysis using PharmLyse (BD) according to the manufacturer’s instructions prior to flow cytometric staining and analysis.
Pharm lyse
Manufactured by BD
Sourced in United States, Germany, United Kingdom, Italy, Belgium
The BD Pharm Lyse is a laboratory reagent used for the lysis of red blood cells in whole blood samples. It is a essential component in the preparation of samples for various hematological analyses.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (21 mentions)
Facscanto 2, by BD (15 mentions)
Facscalibur, by BD (14 mentions)
Lsrfortessa, by BD (13 mentions)
Dnase 1, by Roche (12 mentions)
Dnase 1, by Merck Group (11 mentions)
Facscanto, by BD (10 mentions)
Facsaria 2, by BD (9 mentions)
Fc block, by BD (8 mentions)
Collagenase 4, by Merck Group (7 mentions)
Collagenase type 4, by Worthington (6 mentions)
Facscanto 2 flow cytometer, by BD (6 mentions)
Lsrii flow cytometer, by BD (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Fortessa, by BD (5 mentions)
Facsaria 3, by BD (5 mentions)
Facsdiva, by BD (5 mentions)
Lsr 2 flow cytometer, by BD (5 mentions)
Collagenase dispase, by Roche (5 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (5 mentions)
405 protocols using pharm lyse
1
Isolation and Culture of Primary Endothelial Cells
2
Immune Response Evaluation in Mice
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One week after the last vaccine dose, we euthanized mice and removed the spleen and lungs to assess immune responses. Single-cell suspensions of splenocytes were prepared by gently pressing the cells out of the spleen sac; lysing red blood cells with PharmLyse (BD Pharmingen); washing the cells; and filtering through a 70 µm nylon cell strainer (Falcon). Single-cell suspensions of lung cells were prepared by cutting the lung into small pieces with a scalpel; incubating at 37°C for 1 h with shaking in 10 mL of digestion solution (300 U/mL collagenase type II [Worthington] and 0.15 mg/mL DNase I [Worthington] in PBS); filtering through a 40-µm nylon cell strainer (Falcon); lysing red blood cells with PharmLyse (BD Pharmingen); and washing the cells. Advanced RPMI-1640 (Invitrogen) supplemented with 2% heat-inactivated fetal bovine serum, 2 mM glutamine dipeptide (glutaGRO Supplement, Corning), 10 mM HEPES buffer, 50 µM β-mercaptoethanol, and penicillin (100 IU/mL)-streptomycin (100 µg/mL) was used as the medium.
3
Flow Cytometric Analysis of Circulating Leukocytes
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For flow cytometric analysis of circulating leukocytes, peripheral blood was collected in Na-heparin. Except for the MDSC panel, in which PBMCs were prepared by density gradient centrifugation, erythrocytes were lysed with BD Pharm Lyse. Peripheral blood was washed in Dulbecco’s PBS (DPBS), lysed in 1× BD Pharm Lyse, and washed again in DPBS. PBMC cell suspensions were prepared with Ficoll-Paque following the manufacturer’s protocol. Cells were stored briefly in storage medium (10% heat-inactivated fetal bovine serum/1% L-glutamine/1% pen-strep) before staining with antibody cocktails.
For each panel, one million cells were stained with specific cocktails of fluorochrome-conjugated antibodies (Tables S3 and S4). Cells were washed with DPBS and then stained with dead cell dye (BD Fixable Viability Stain 700) before washing with wash buffer (0.5% BSA/DPBS/NaN3). Cells were then treated with 50 μl of Fc-blocking solution (2% normal rabbit serum/10% BD Fc Block/DPBS) before application of a 100-μl antibody cocktail diluted in wash buffer. Samples were stained within 6 h of sample collection and analyzed on a BD Biosciences FACSCanto flow cytometer within 2 h of staining. The stopping gate was set to acquire 500,000 viable, nucleated single cells.
For each panel, one million cells were stained with specific cocktails of fluorochrome-conjugated antibodies (Tables S3 and S4). Cells were washed with DPBS and then stained with dead cell dye (BD Fixable Viability Stain 700) before washing with wash buffer (0.5% BSA/DPBS/NaN3). Cells were then treated with 50 μl of Fc-blocking solution (2% normal rabbit serum/10% BD Fc Block/DPBS) before application of a 100-μl antibody cocktail diluted in wash buffer. Samples were stained within 6 h of sample collection and analyzed on a BD Biosciences FACSCanto flow cytometer within 2 h of staining. The stopping gate was set to acquire 500,000 viable, nucleated single cells.
4
Comprehensive Profiling of NSCLC Tumor Microenvironment
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NSCLC tumor tissue, matched normal lung tissue, and peripheral blood were obtained from 39 patients between May 2015 and October 2016.
White blood cells were extracted from peripheral blood by lysis with BD Pharm lyse (BD Biosciences) red blood cell lysis buffer and then subjected to a FACS analysis. PBMC were isolated from peripheral blood by gradient density centrifugation using Lymphoprep (Axis Shield), and this was followed by a T-cell functional analysis.
Fresh tumor or normal lung tissues were minced in a 6-cm dish and digested to a single cell suspension using a Tumor Dissociation Kit for humans (Miltenyi Biotec) and gentleMACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. The cell suspension was applied to a 70-μm nylon cell strainer (BD Biosciences) with the lysis of red blood cells by BD Pharm lyse. Dead cells and debris were removed by centrifugation in isodensity Percoll solution (Pharmacia Biotech), followed by FACS and T-cell functional analyses. The remaining cells were cryopreserved in liquid nitrogen for the TCR repertoire analysis.
White blood cells were extracted from peripheral blood by lysis with BD Pharm lyse (BD Biosciences) red blood cell lysis buffer and then subjected to a FACS analysis. PBMC were isolated from peripheral blood by gradient density centrifugation using Lymphoprep (Axis Shield), and this was followed by a T-cell functional analysis.
Fresh tumor or normal lung tissues were minced in a 6-cm dish and digested to a single cell suspension using a Tumor Dissociation Kit for humans (Miltenyi Biotec) and gentleMACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. The cell suspension was applied to a 70-μm nylon cell strainer (BD Biosciences) with the lysis of red blood cells by BD Pharm lyse. Dead cells and debris were removed by centrifugation in isodensity Percoll solution (Pharmacia Biotech), followed by FACS and T-cell functional analyses. The remaining cells were cryopreserved in liquid nitrogen for the TCR repertoire analysis.
5
Circulating Leukocyte Analysis by Flow Cytometry
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For flow cytometric analysis of circulating leukocytes, peripheral blood
was collected in Na-heparin. Except for the MDSC panel, in which PBMCs were
prepared by density gradient centrifugation, erythrocytes were lysed with BD
Pharm Lyse. Peripheral blood was washed in Dulbecco’s PBS (DPBS), lysed
in 1× BD Pharm Lyse, and washed again in DPBS. PBMC cell suspensions were
prepared with Ficoll-Paque following the manufacturer’s protocol. Cells
were stored briefly in storage medium (10% heat-inactivated fetal bovine
serum/1% L-glutamine/1% pen-strep) before staining with antibody cocktails.
For each panel, one million cells were stained with specific cocktails
of fluorochrome-conjugated antibodies (Supplementary Table 3-4). Cells were washed
with DPBS then stained with dead cell dye (BD Fixable Viability Stain 700)
before washing with wash buffer (0.5% BSA/DPBS/NaN3). Cells were then
treated with 50 μL of Fc-blocking solution (2% normal rabbit serum/10% BD
Fc Block/DPBS)before application of a 100-μL antibody cocktail diluted in
wash buffer. Samples were stained within 6 hours of sample collection and
analyzed on a BD Biosciences FACSCanto flow cytometer within 2 hours of
staining. The stopping gate was set to acquire 500,000 viable, nucleated single
cells.
was collected in Na-heparin. Except for the MDSC panel, in which PBMCs were
prepared by density gradient centrifugation, erythrocytes were lysed with BD
Pharm Lyse. Peripheral blood was washed in Dulbecco’s PBS (DPBS), lysed
in 1× BD Pharm Lyse, and washed again in DPBS. PBMC cell suspensions were
prepared with Ficoll-Paque following the manufacturer’s protocol. Cells
were stored briefly in storage medium (10% heat-inactivated fetal bovine
serum/1% L-glutamine/1% pen-strep) before staining with antibody cocktails.
For each panel, one million cells were stained with specific cocktails
of fluorochrome-conjugated antibodies (Supplementary Table 3-4). Cells were washed
with DPBS then stained with dead cell dye (BD Fixable Viability Stain 700)
before washing with wash buffer (0.5% BSA/DPBS/NaN3). Cells were then
treated with 50 μL of Fc-blocking solution (2% normal rabbit serum/10% BD
Fc Block/DPBS)before application of a 100-μL antibody cocktail diluted in
wash buffer. Samples were stained within 6 hours of sample collection and
analyzed on a BD Biosciences FACSCanto flow cytometer within 2 hours of
staining. The stopping gate was set to acquire 500,000 viable, nucleated single
cells.
6
Spleen Dissection and Splenocyte Isolation
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Mice were euthanized and the spleens dissected and stored on ice. Splenocytes were then isolated and purified through a 40 μm mesh filter. The splenocytes were collected by centrifugation at 4°C at 500 × g for 5 min. For each spleen, the supernatant was discarded and the pellet dissolved in 1 ml 1× Pharm Lyse (BD Biosciences) and the samples were incubated at room temperature for 3 min to lyse splenic erythrocytes. To terminate the lysis reaction, 0.5 ml phosphate buffered saline (PBS) was added and the splenocytes were collected by centrifugation at 4°C, 500 × g for 5 min, the supernatant discarded and the pellet resuspended in 0.5 ml PBS. After one final centrifugation step for 2 min, the supernatant was discarded and cell pellet resuspended in 1 ml freeze medium (PBS with 10% Dimethyl Sulfoxide and 10% fetal calf serum). The samples were stored at minus 80°C until TLA processing.
7
Immune Cell Profiling by Flow Cytometry
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Erythrocytes were lysed using 1× PharmLyse (BD Biosciences Franklin Lanes, NJ, USA) according to the manufacturer’s instructions. Cells were washed and resuspended in PBS pH 7.4, containing 2% FBS and 0.09% sodium azide (stain buffer). An amount of 5 × 105 cells were stained for 30 min on ice with immune cell-specific antibodies (BD Biosciences, Franklin Lakes, NJ, USA) as follows: CD8a (#561611), CD4 (#554837), CD3 (#554833), CD11b/c (#743980), CD161a (#555009), or CD45RA (#554881) at a concentration of 1:100 diluted in stain buffer. Cells were washed two times with 2 mL stain buffer and centrifuged at 350× g for 5 min at 4 °C. Cells were resuspended in 400 μL of stain buffer and immediately analyzed using a BD FACSymphony A3 Flow Cytometer (BD Biosciences, Franklin Lanes, NJ, USA) at the UMMC Flow Cytometry and Cell Sorting Core Facility. Data were analyzed using FlowJo software version 10.8.2. Gating strategy for both the brain and PBL are included in Supplemental Figure S1 .
8
Engrafting NSG Mice with Human Hematopoietic Stem Cells
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Studies were conducted at Fred Hutch Cancer Research Center under approval of the Institutional Animal Care and Use Committee (IACUC) protocol Proto202000003 (online supplemental table S3 ). Female Nod.Cg-PrkdcscidIL2rgtm1Wjl/SzJ (NSG) mice were irradiated and engrafted with CD34+ human hematopoietic stem cells at Jackson Laboratories (Bar Harbor, Maine, USA). UB-VV100 formulation was thawed on the day of treatment, diluted in PBS, and injected via intraperitoneal injection. Blood was collected via retroorbital sinus into sodium–EDTA coated microtainers and centrifuged at 4000×g for 10 min to collect plasma. Blood was lysed in 1× Pharmlyse (BD Biosciences, San Diego, California, USA) and washed in PBS (Life Technologies, Rockville, Maryland, USA). Cells were stained for viability and surface antibody (online supplemental table S1 ). Samples were acquired on a 4-laser Cytoflex S (Beckman Coulter) and analyzed using FlowJo V.10 (Ashland, Oregon, USA). Data were graphed in Microsoft Excel and plotted on GraphPad Prism V.9 (San Diego, California, USA).
9
Isolation and Characterization of Adipose Tissue Macrophages
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Mice were injected intraperitoneally with 3% sodium thioglycollate (Wako). After four days, peritoneal MΦs were collected and stimulated with IL-4. Epididymal adipose tissue specimens isolated from mice were rinsed in PBS, minced into fine pieces, and digested with Tyrode buffer (130 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 0.33 mM NaH2PO4, 22 mM glucose, 5 mM L-glutamine, and 25 mM HEPES) containing collagenase (Worthington) at 37 °C using a water bath for 15 min. Then, the samples were passed through a mesh and centrifuged in a swing rotor at 362 × g. The pellets were collected as the stromal-vascular fraction (SVF), and the SVF cells were incubated in 1× Pharm Lyse (BD Biosciences) for 8 min at room temperature. The cells were suspended in PBS containing 2% BSA, and incubated with anti-CD16/32 (553142, BD pharmingen) for 5 min on ice. Then, the cells were incubated with the primary antibodies or the matching control isotypes for 1 h on ice, and analyzed using a FACS Aria II cell sorter (BD Biosciences) and FlowJo (Tree Star, Ashland, OR). SiglecF-negative/F4/80-positive/CD11b-positive cells were sorted and used for the RNA extraction. Furthermore, the cells were also divided CD11c-positive/CD206-negative and CD11c-negative/CD206-positive cells, as M1-type and M2-type MΦs, respectively.
10
Multicolor Flow Cytometry of Bone Cells
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Multicolour flow cytometric analysis of Nestin+, endothelial and osteoblast compartments was performed at 2 months of age as previously reported42 (link). In brief, Nestin+ mesenchymal cells and other stromal cell populations were isolated from femur and tibia. Bones were crushed in a mortar with a pestle and digested with 2 ml of collagenase (Stem Cell Technologies, 07092) and 1 mg/ml collagenase IV (Sigma) at 37 °C in strong agitation for 30 min. Cells were then filtered through a 40 µm mesh and red blood cells lysed with 1× Pharmlyse (BD) for 10 min on ice.
Apoptosis was detected using the Annexin-V Apoptosis Detection Kit I (eBioscience, Winsford, UK). Samples were acquired on a LSRII flow cytometer (BD Biosciences, Oxford, UK) and analysed using FlowJo software (Tree Star).
Antibodies for fluorescence-activated cell sorting analysis are listed in Supplementary Table6 .
Apoptosis was detected using the Annexin-V Apoptosis Detection Kit I (eBioscience, Winsford, UK). Samples were acquired on a LSRII flow cytometer (BD Biosciences, Oxford, UK) and analysed using FlowJo software (Tree Star).
Antibodies for fluorescence-activated cell sorting analysis are listed in Supplementary Table
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