Primary AML leukaemia cells were isolated from the bone marrow of AML patients at diagnosis from the Second Hospital of Anhui Medical University. Briefly, bone marrow blood was slowly layered over Ficoll‐Paque PLUS solution (GE Healthcare Life Sciences, Sweden) and centrifuged at 550×g for 25 mins. The mononuclear cells in the interphase layer were carefully transferred into another fresh tube, washed with Hank's balanced salt solution (Beyotime Biotechnology) twice and then applied to subsequent experiments. CD34+ haematopoietic stem/progenitor cells (HSPCs) and CD34− cells were purified from the cord blood of healthy donors from the First Affiliated Hospital of Anhui Medical University using Ficoll‐Paque PLUS and anti‐CD34‐coated magnetic beads (Miltenyi Biotec). Briefly, we isolated mononuclear cells from fresh cord blood as described above. Next, a single cell suspension obtained was incubated with anti‐CD34‐coated beads and selected on autoMACS Pro (Miltenyi Biotec). CD34+ cells were identified by incubation with CD34 antibody (BioLegend, USA) and analysis with flow cytometry (CytoFLEX, Beckman Coulter).
Ficoll paque plus
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
Ficoll‐Paque PLUS is a density gradient medium used for the isolation of mononuclear cells from whole blood or bone marrow. It is a sterile, pyrogen-free solution of Ficoll and sodium diatrizoate in water, with a density of 1.077 g/mL.
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Lab products found in correlation
Cd34 beads, by Miltenyi Biotec (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Nycoprep 1.077a, by Axis-Shield (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus solution, by GE Healthcare (1 mentions)
Hanks balanced salt solution (hbss), by Beyotime (1 mentions)
Anti cd34 coated magnetic beads, by Miltenyi Biotec (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Cd34 antibody, by BioLegend (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
M csf, by BD (1 mentions)
Human cd16 microbeads, by Miltenyi Biotec (1 mentions)
Trizol, by Roche (1 mentions)
Macs positive selection, by Miltenyi Biotec (1 mentions)
Apc cy7 anti human cd4 antibody, by BioLegend (1 mentions)
Pe cy7 antihuman cd3 antibody, by BioLegend (1 mentions)
Pe mouse anti human cd158a, by BD (1 mentions)
Pe mouse anti human cd158b, by BD (1 mentions)
Percp cyanine5.5 anti human cd19, by BioLegend (1 mentions)
Fcr cd16 32 blocking reagent, by Miltenyi Biotec (1 mentions)
8 protocols using ficoll paque plus
1
Isolation of AML and Healthy HSPCs
2
Culturing and Differentiating THP-1 and PBMC-derived Macrophages
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THP-1 cells were cultured in RPMI-1640 supplied with 10% FBS, 1% penicillin, and streptomycin in a 5%-CO2 humidified incubator at 37°C. The THP-1 cells were stimulated with 10 nM PMA for 48 h, then THP-1-derived macrophages were differentiated. The PBMC-derived macrophages were cultured and differentiated as previously reported (24 (link)). Briefly, human PBMCs were separated by centrifugation on Ficoll-Paque Plus and purified by CD14-positive cells isolation kit (Miltenyi Biotec, CA, USA). The purified cells were differentiated in complete RPMI-1640 supplied with M-CSF (10 ng/ml) (BD Biosciences, CA, USA) for 6 days. Donor blood samples were randomly collected in the Jiangsu Province Blood Center. The study was approved by Ethical Committee of Anhui Medical University Affiliated with Bayi Clinical College and all participants signed an informed consent form when they filled the questionnaire.
3
Isolation and RNA Extraction of Human Neutrophils
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Human neutrophils were isolated from venous blood of patients and healthy controls by density gradient centrifugation using Ficoll-Paque Plus according to the manufacturer's protocol and then followed by positive magnetic separation for further purification using human CD16 Microbeads (Miltenyi Biotec). The cells were dissolved in TRIzol (Roche, America) in a volume of 5 − 10 × 106 cells/1 mL, followed by storage at -80°C. Total RNA was extracted, and the concentration and purity of RNA were tested on a NanoDrop spectrophotometer, followed by reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA).
4
Purification of Leukemic and Hematopoietic Cells
5
Isolation of CD4+ T-cells from Blood
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Blood samples drawn on anticoagulant-containing tubes (Sarstedt, Nümbrecht, Germany) were initially twice diluted with PBS (without Ca2+ and Mg2+). Peripheral blood mononuclear cells (PBMC) were separated from whole blood on a density gradient (Ficoll-paque plus); next, the CD4-positive T-cells were isolated using magnetic assay cell sorting (MACS positive selection, Miltenyi Biotec, Teterow, Germany). The cell number was determined by cell counting in a Bürker chamber.
6
Immunophenotyping of Peripheral Blood and Bone Marrow Cells
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PB and BM samples from HDs and AA patients were collected in EDTA anticoagulant tubes and stored at 4 °C. PBMCs and BMMCs were isolated by density gradient centrifugation using Ficoll‐Paque PLUS within 24 h. PBMCs and BMMCs were blocked using FcR (CD16/32) Blocking Reagent (Miltenyi), and stained with the following antibodies/reagents: PE anti‐human CD158f (KIR2DL5) antibody (Biolegend), PE anti‐human CD158e1 (KIR3DL1, NKB1) antibody, PE mouse anti‐Human CD158a (BD Pharmingen), PE mouse anti‐human CD158b (BD Pharmingen), APC anti‐human TCRγ/δ (Biolegend), Biotin anti‐humanTCRvδ2 (Biolegend), APC/Cyanine7 streptavidin (Biolegend), human KIR3DL2/CD158k PE‐conjugated antibody (R&D), anti‐humanCD261(DR4)‐APC (Miltenyi), FITC anti‐human CD8a antibody (Biolegend), APC‐Cy7 anti‐human CD4 antibody (Biolegend), Percp‐Cy5.5 anti‐human CD25 antibody (Biolegend), PE‐Cy7 anti‐human CD127 antibody (eBioscience), PE/Cyanine7 Streptavidin (Biolegend), PerCP/Cyanine5.5 anti‐human CD19 (BioLegend), PE‐Cy7 anti‐human CD3 antibody (BioLegend), APC anti‐human TCRγ/δ antibody (BioLegend), and PE anti‐human CD184 (CXCR4) antibody (BioLegend). The cells were resuspended in 400 µl 0.1 µg mL−1 DAPI solution (Salarbio). Treg cells (CD8a−CD4+CD127−/lowCD25+) were sorted using an Arial II (BD Biosciences).
7
Purification of Leukemic and Normal Cells
8
Humanized Mice for Cancer Immunotherapy
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Human umbilical cord blood-derived mononuclear cells were purified from healthy donors by density-gradient centrifugation using Ficoll Paque PLUS at a density of 1.068 g/mL (Miltenyi Biotec). CD34 + stem cells were extracted from cord blood-derived mononuclear cells using CD34 MicroBead Kit UltraPure human (Miltenyi Biotec). To obtain CD34 + humanized mice, 4-to 5-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were treated with 1.5 Gy gamma radiation 12-24 hours before implantation of 5 × 10 4 freshly isolated CD34 + cells via tail vein. Engraftment of human immune cells was monitored by means of flow cytometry, with >25% human CD45 + cells in each mouse. After 16 weeks, HCT116 cells with single guide RNA negative control and ALKBH5-KO were subcutaneously injected into CD34 + humanized mice (1 × 10 7 cells/mouse). Tumor size and immune cell infiltration were analyzed. Additional methods are provided in the Supplementary Material.
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