The stromal vascular fraction (SVF) of iBAT and iWAT were isolated as previously described.[44 ] In brief, the adipose tissue was minced and then incubated in working solution (1.5 mg mL−1 collagenase type II, 2.4 U mL−1 dispase II, and 10 mm CaCl2) for 30 min at 37 °C. The cell suspension was then filtered and centrifuged. The suspended SVF cells were then incubated in erythrocyte‐lysing buffer for 5 min, spun down by centrifugation, and re‐suspended in PBS containing 5% fetal bovine serum (FBS). The following antibodies (at 1:100) were used to analyze the macrophages (all from Biolegend): anti‐CD11b (M1/70), anti‐F4/80 (BM8), and anti‐CD11c (N418). For analyzing stem cells, anti‐CD34 antibody (Biolegend, RAM34) was used. Labeled cells were analyzed using an ACEA NovoCyte flow cytometer (BD Biosciences). FACS data were analyzed using FlowJo (Tree Star, Inc., Ashland, OR). The gating map for analyzing macrophages is shown in Figure S3G, Supporting Information. The ratio of macrophages to total cells and inflammatory CD11c+ cells to total macrophages were calculated.
Anti cd34 antibody
Manufactured by BioLegend
Sourced in United States
The Anti-CD34 antibody is a laboratory reagent used to detect and identify cells expressing the CD34 antigen. CD34 is a transmembrane phosphoglycoprotein that is expressed on hematopoietic stem and progenitor cells. The antibody can be used in various applications, such as flow cytometry, immunohistochemistry, and cell sorting, to study and characterize CD34-positive cell populations.
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Lab products found in correlation
Anti cd45 antibody, by BioLegend (2 mentions)
Anti cd3 antibody, by BioLegend (2 mentions)
Anti cd11c n418, by BioLegend (1 mentions)
Acea novocyte flow cytometer, by BD (1 mentions)
Fcr blocker, by Miltenyi Biotec (1 mentions)
Dioc6, by Thermo Fisher Scientific (1 mentions)
Anti cd19 antibody, by BioLegend (1 mentions)
Anti cd123, by BioLegend (1 mentions)
Anti cd49b antibody, by BioLegend (1 mentions)
Anti ter 119 antibody, by BioLegend (1 mentions)
Anti tcrβ antibody, by BioLegend (1 mentions)
Anti cd11c antibody, by BioLegend (1 mentions)
Anti cd11b antibody, by BioLegend (1 mentions)
Anti ly6g antibody, by BioLegend (1 mentions)
Facsaria, by BD (1 mentions)
Rbc lysis buffer, by BD (1 mentions)
Anti cd105 antibody, by R&D Systems (1 mentions)
Facscan flow cytometer, by Beckman Coulter (1 mentions)
Cellquest analysis software, by BD (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti cd34 antibody
1
Isolation and Characterization of Adipose Tissue SVF
2
Galectin-9 Effect on CD34+ AML Cells
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DiOC6 (Molecular Probes, Eugene, Oregon, USA) at a concentration of 0.1 µM in fresh culture medium was added 1:1 to cells. After 20 min of incubation at 37 °C, cells were washed with PBS, and collected by centrifugation (450 g, 5 min), resuspended in PBS, and analyzed using flow cytometry.
To determine the percentage of CD34+ AML cells after treatment with Gal-9, AML cells were pre-incubated with FcR-blocker (100μg/mL) (Miltenyi Biotec, Leiden, The Netherlands) and subsequently stained with anti-CD34 antibody ((Clone: 561, Biolegend, California, USA) for 1 h at 4oC. Cells were washed with PBS and collected by centrifugation (450g, 5 min), resuspended in PBS, and analyzed using flow cytometry.
To determine the percentage of CD34+ AML cells after treatment with Gal-9, AML cells were pre-incubated with FcR-blocker (100μg/mL) (Miltenyi Biotec, Leiden, The Netherlands) and subsequently stained with anti-CD34 antibody ((Clone: 561, Biolegend, California, USA) for 1 h at 4oC. Cells were washed with PBS and collected by centrifugation (450g, 5 min), resuspended in PBS, and analyzed using flow cytometry.
3
Characterization of Immune Landscape in 4T1 Tumor Model
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The livers and bone marrow cells were harvested from sham and 4T1 cancer-bearing animals 14 days after transplantation. Red blood cells (RBCs) were lysed with RBC lysis buffer (BD Bioscience). The obtained cells were then stained with the following antibodies (BioLegend, CA, USA) for 30 min at 4 °C: anti-CD45 antibody (Clone: 30-F11), anti-CD19 antibody (Clone: 6D5), anti-TCRβ antibody (Clone: H57-597), anti-CD11b antibody (Clone: M1/70), anti-Ly6G antibody (Clone: 1A8), anti-FCεR1a antibody (Clone: MAR-1), anti-CD117 antibody (Clone: 2B8), anti-CD123 (Clone: 5B11), anti-CD49b antibody (Clone: DX5), anti-CD3 antibody (Clone: 17A2), anti-CD11c antibody (Clone: N418), anti-TER-119 antibody, or anti-CD34 antibody (Clone: HM34). Non-viable cells were stained with Propidium Iodide Solution (PI) and gated out. Data were acquired using FACS Aria (BD Bioscience) and analyzed using FlowJo software (v10.8.1, BD Biosciences). Gating strategy is shown in Supplementary Fig. 14 .
4
Rat Adipose-Derived Stem Cell Characterization
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ADSCs of rats were digested by 0.25% trypsinase for 15 minutes at 37°C. After being washed twice with PBS, the cells were incubated with monoclonal antibodies, including anti-CD29 antibody (BioLegend, San Diego, CA, USA), anti-CD90 antibody (BioLegend), anti-CD105 antibody (R&D Systems, Inc., Minneapolis, MN, USA), anti-CD45 antibody (BioLegend), anti-CD106 antibody (BioLegend), or anti-CD34 antibody (BioLegend). The secondary antibodies, antirabbit or goat fluorescein isothiocyanate-conjugated antibodies (BD, Franklin Lakes, NJ, USA), were used according to the manufacturer’s instructions. Negative controls were conducted by omitting the primary antibodies. The scatter parameters of ADSCs were analyzed using FACScan flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA) and CellQuest analysis software (BD).
5
PBMC Isolation and CAR-T Cell Generation
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Density-gradient centrifugation was used to isolate PBMCs from healthy donor samples or patients with MM by Ficoll-Paque (General Electric). T cells were isolated from PBMCs with anti-CD3 microbeads (Miltenyi Biotec, 130-050-101) and were cultured in AIM V Medium (Thermo Fisher Scientific) with 5% human AB serum (MilliporeSigma) and 400 IU IL-2 (R&D Systems). Dynabeads of human T-activator CD3/CD28 (Thermo Fisher Scientific, 11161D) were added for T cell expansion and activation (26 (link)). T cells were activated for 2–3 days prior to transduction.
Lentivirus particles were used to transduce T cells. T cells and concentrated lentiviruses were added into RetroNectin-precoated plates (Takara Bio) (26 (link)). Cells were cultured in AIM V Media for 24 hours, and the transduction step was repeated. After 24 hours, cells were washed with PBS and cultured in fresh media for 7 days. CAR-T cells were detected by BD FACSVerse flow cytometry with anti-CD34 antibodies (BioLegend, clone: 561, no. 343606), anti-CD3 antibodies (BioLegend, clone: HIT3a, no. 300318), and CD34 microbeads (Miltenyi Biotec, 130-046-703) for isolation. CD4 or CD8 phenotypes of CAR-T cells were detected by flow cytometry using anti-CD4 antibodies (BioLegend, clone: RPA-T4, no. 300508) and anti-CD8 antibodies (BioLegend, clone: SK1, no. 344722).
Lentivirus particles were used to transduce T cells. T cells and concentrated lentiviruses were added into RetroNectin-precoated plates (Takara Bio) (26 (link)). Cells were cultured in AIM V Media for 24 hours, and the transduction step was repeated. After 24 hours, cells were washed with PBS and cultured in fresh media for 7 days. CAR-T cells were detected by BD FACSVerse flow cytometry with anti-CD34 antibodies (BioLegend, clone: 561, no. 343606), anti-CD3 antibodies (BioLegend, clone: HIT3a, no. 300318), and CD34 microbeads (Miltenyi Biotec, 130-046-703) for isolation. CD4 or CD8 phenotypes of CAR-T cells were detected by flow cytometry using anti-CD4 antibodies (BioLegend, clone: RPA-T4, no. 300508) and anti-CD8 antibodies (BioLegend, clone: SK1, no. 344722).
6
Isolation and Culture of CD34+ HSPCs
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CD34+ hematopoietic stem/progenitor cells (HSPCs) were sorted from the cord blood of healthy donors from the First Affiliated Hospital of Anhui Medical University using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Stockholm, Sweden) and anti-CD34-coated magnetic beads (Miltenyi Biotec, Bergishgradbach, Germany). Mononuclear cells were isolated from fresh cord blood as previously described. A single-cell suspension was incubated with anti-CD34-coated beads and selected on MACS (Miltenyi Biotec, Bergishgradbach, Germany). The purity of CD34+ cells was analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Miami, FL, USA) following incubation with anti-CD34 antibodies (BioLegend, San Diego, CA, USA). Primary CD34+ HSPCs were cultured in StemSpan SFEM medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 2 mmol/L L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% Lipid Mixture 1 (L0288, Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL SCF (Human origin, PeproTech, Rocky Hill, CT, USA), 2 ng/mL IL-3 (Human origin, PeproTech, Rocky Hill, CT, USA), and 1% penicillin-streptomycin.
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