K562 cells (no. SCSP-5054) and 293 T cells (no. SCSP-502) were obtained from the Cell Bank of the Chinese Academy (www.cellbank.org.cn ). K562 cells were maintained in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), and 293 T cells were maintained in Dulbecco’s modified Eagle medium (DMEM) plus 10% FBS. Murine BaF3 cells (from Dr. Connie J. Eaves, Terry Fox Laboratory, the British Columbia Cancer Research Institute) were maintained in RPMI 1640 medium supplemented with 10% FBS and 5 ng/mL mIL-3. Murine BaF3-BCR/ABL cells were generated by BCR/ABL lentiviral transduction and were maintained in RPMI 1640 medium plus 10% FBS. Bone marrow cells (BMCs) from human CML patients and healthy donors were obtained from the Hematological Biobank, Jiangsu Biobank of Clinical Resources. Nucleated cells (unfractioned BMCs) were obtained using a gradient centrifuge with lympholyte-H cell separation media (Cedarlane Laboratories, Burlington, NC, USA), and CD34+ cells were purified using an EasySep CD34 positive selection kit (STEMCELL Technologies, Vancouver, BC, Canada). The clinical characteristics of CML patients recruited in the present study are summarized in Additional file 1 : Table S1.
Lympholyte h cell separation media
Manufactured by Cedarlane
Sourced in Canada, United States
Lympholyte®-H Cell Separation Media is a density gradient medium used for the isolation and separation of mononuclear cells from whole blood or other cell suspensions.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Easysep cd34 positive selection kit, by STEMCELL (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
K2edta tubes, by BD (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Genelute blood genomic dna kit, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd8 microbead, by Miltenyi Biotec (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Mir x mirna qrt pcr sybr kit, by Takara Bio (1 mentions)
Abi 7500 real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Dimethylsulfoxyde dmso, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
15 protocols using lympholyte h cell separation media
1
Cell Line and Primary Cell Culture Protocol
2
Isolation and Cryopreservation of PBMCs
3
CMV-Seropositive Donor PBMC Protocol
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A total number of 73 CMV-seropositive donors, aged between 24–88 years, with appropriate HLA-genotype were included in the study. PBMCs were isolated from 50ml heparinized blood by density gradient centrifugation using Lympholyte-H cell separation media (Cedarlane) and aliquots of 10x106 cells were cryopreserved in RPMI1640 (Sigma-Aldrich) containing 20% fetal calf serum (FCS) and 10% DMSO and stored in liquid nitrogen until use.
To identify donors with the appropriate HLA-genotype, genomic DNA was isolated from PBMCs using the GenElute Blood Genomic DNA Kit (Sigma-Aldrich) according to manufacturer’s instructions. Typing for HLA class II alleles was performed by PCR technique as described previously [72 (link)].
To identify donors with the appropriate HLA-genotype, genomic DNA was isolated from PBMCs using the GenElute Blood Genomic DNA Kit (Sigma-Aldrich) according to manufacturer’s instructions. Typing for HLA class II alleles was performed by PCR technique as described previously [72 (link)].
4
Mouse and Human Immune Cell Isolation
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Mouse splenocytes were isolated as described in (Birrell et al., 2005 (link)). Human peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers using Lympholyte-H Cell Separation Media (Cedarlane Labs, Canada), according to manufacturer’s instruction and in accordance with local ethical guidance.
Splenocytes (following red blood cell lysis) or cell lines were washed in PBS containing 1% fetal calm serum (flow buffer) and then re-suspended in 10 μg/ml Fc Block for 15 min on ice. Cells were subsequently washed and re-suspended in flow buffer containing primary antibody (Ab) (0.1–3 μg/ml) for 30 min on ice. Cells were then washed 3 times in flow buffer and incubated with the appropriate secondary reagent for 30 min. Cells were washed and resuspended in flow buffer containing 1% formaldehyde. Samples were analysed using a FACS Canto ™ II (BD, Oxford, UK) and FlowJo (LLC, Ashland, OR, USA) or Cyflogic (v 1.2.1, free ware).
Splenocytes (following red blood cell lysis) or cell lines were washed in PBS containing 1% fetal calm serum (flow buffer) and then re-suspended in 10 μg/ml Fc Block for 15 min on ice. Cells were subsequently washed and re-suspended in flow buffer containing primary antibody (Ab) (0.1–3 μg/ml) for 30 min on ice. Cells were then washed 3 times in flow buffer and incubated with the appropriate secondary reagent for 30 min. Cells were washed and resuspended in flow buffer containing 1% formaldehyde. Samples were analysed using a FACS Canto ™ II (BD, Oxford, UK) and FlowJo (LLC, Ashland, OR, USA) or Cyflogic (v 1.2.1, free ware).
5
Isolation of Immune Cell Subsets
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These assays were approved by Shanghai Public Health Clinical Center and Shanghai Changhai Hospital. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood by density gradient centrifugation using Lympholyte®-H Cell Separation Media (Cedarlane Laboratories) as previously described13 (link),27 (link). CD4+ T cells were isolated from PBMCs by negative selection using CD4+ T cell Isolation Kit (Miltenyi Biotech). Resting CD4+ T cells were isolated from CD4+ T cells by depletion of cells expressing CD69, CD25 or HLA-DR using CD25-Biotin, CD69-Biotin and HLA-DR-Biotin antibody-coated magnetic beads (Miltenyi Biotech). CD8+ T cells were isolated from PBMCs by positive selection using CD8 MicroBeads (Miltenyi Biotech).
6
Profiling miR-17-92 Cluster in PBMCs
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PBMCs were isolated from peripheral blood of the IS group and the control group using a commercial kit (Lympholyte®-H Cell Separation Media, Cedarlane). Total RNA was extracted from PBMCs of the two groups using a commercial kit (Takara, Dalian, China). For examination of the miR-17-92 cluster, 3.75 μl of RNA (0.25–8 μg) was converted to cDNA using a commercial kit according to the manufacturer’s manual (Mir-X miRNA First-Strand Synthesis Kit, Takara; Cat.No.638315). Commercial primer sets of miR-17-92 cluster were obtained from Shanghai Genesky Biotechologies Inc. Quantitative reverse transcription PCR was performed using Mir-X miRNA qRT-PCR SYBR Kit (Takara, Cat. No.638313) and ABI 7500 real-time PCR machine (Applied Biosystems, CA, USA). Data were normalized using U6 snRNA as an internal control, and the relative expression of the miR-17-92 cluster was quantified using delta-delta Ct (2-ΔΔCt) method.
7
Isolation of PBMCs from Whole Blood
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We isolated peripheral blood mononuclear cells (PBMCs) from whole blood Ethylene Diamine Tetraacetic Acid (EDTA) samples by density gradient centrifugation method (Lympholyte®-H Cell Separation Media, CEDARLANE). PBMC pellets were washed and resuspended in Dulbecco’s phosphate-buffered saline (DPBS) (GIBCO, Invitrogen) and aliquoted according to the final analyses.
8
Isolation and Purification of Hematopoietic Stem Cells
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K562, MEG-01 and SW620 cells were purchased from the cell bank of Chinese Academy (www.cellbank.org.cn ), which were maintained with RPMI1640 plus 10% FBS. The primary CML or normal adult bone marrow samples were collected with informed consent forms in the Department of Hematology, the First Affiliated Hospital, Soochow University. The clinical characteristics of these patients were summarized in Table S1 in File S1 . After gradient centrifuge with Lympholyte®-H cell separation media (Cedarlane Laboratories, Burlington, NC, USA), the nucleated cells were yielded and then purified with human CD34 EasySep™ kit (Stem Cell Technologies, Vancouver, BC, Canada) following the instruction of the manufacturer.
9
Cryopreserved PBMC Isolation from Septic and Healthy Blood
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PBMC from septic patients and healthy volunteers were isolated from 7 mL of blood freshly drawn on anticoagulated tubes with Lympholyte®-H Cell Separation Media (Cedarlane, Burlington, Canada) centrifugation. PBMC were immediately cryoconserved after isolation in Roswell Park Memorial Institute medium 1640 (RPMI) complemented with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and glutamine (Eurobio Abcys, Courtaboeuf, France) containing 40% AB human serum (SAB) and 10% dimethylsulfoxyde (DMSO) (Sigma-Aldrich, Saint-Louis, MO).
10
Isolation of PBMCs from Peripheral Blood
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Of the 34 patients who underwent LSG, 23 patients who consented to the collection of additional blood samples at the time of LSG were included in the analysis. Mononuclear cells were extracted from peripheral blood immediately after blood sampling. The blood specimens were diluted with phosphate-buffered saline, and peripheral blood mononuclear cells (PBMCs) were separated by standard density gradient centrifugation using Lympholyte®-H cell separation media, Human (Cedarlane Laboratories Ltd., Ontario, Canada) at 1500 rpm for 30 minutes. After the collection of PBMCs at interphase and washing with phosphate-buffered saline, the cell numbers were determined.
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