Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient (Axis-Shield, Oslo, Norway) and cryopreserved in freezing medium (10% DMSO, 90% fetal calf serum; Lonza, CA, USA) in liquid nitrogen until used. PBMCs were thawed, washed twice with phosphate-buffered saline (PBS, Lonza, CA, USA), and stained at room temperature for 15 min with the following antibodies: BV570-CD3 (clone UCHT1), APC-Cy7-CD4 (clone A161A1), and Alexa Fluor 700-CD8 (clone RPA-T8) (BD Biosciences, San Jose, CA, USA). Viability was determined using live/dead aqua fluorescent reactive dye (Thermo Fisher Scientific, CA, USA). Cells were washed and fixed in 300 μl of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and acquired immediately in a FACS Symphony cytometer (BD, Biosciences). Fluorescence minus one stained tubes were used as gating controls. Data were analyzed using FlowJo v.10 software (FlowJo LLC, Ashland, OR, USA) (Supplementary Figure S1 ).
Ficoll density gradient
Manufactured by Axis-Shield
Sourced in Norway
Ficoll density gradient is a laboratory technique used for the separation and purification of cells, organelles, or other biological macromolecules. It utilizes a gradient of Ficoll, a highly branched polysaccharide, to create distinct density layers that allow the target material to be isolated based on its specific density.
Automatically generated - may contain errors
Lab products found in correlation
Facs symphony cytometer, by BD (1 mentions)
Live dead aqua fluorescent reactive dye, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Macs cd14 microbeads, by Miltenyi Biotec (1 mentions)
Cd1c bdca1 dc isolation kit, by Miltenyi Biotec (1 mentions)
Pkh26, by Merck Group (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
4 protocols using ficoll density gradient
1
Cryopreserved PBMC Immunophenotyping
2
Isolation and Sorting of Leukemia Stem Cells
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Mononuclear cells were isolated by Ficoll density gradient (Axis-Shield Diagnostics Ltd., Dundee, Scotland), complemented by CD34 isolation if the CD34 expression was <50% (CD34 MicroBead Kit, Milteny Biotec B.V., Leiden, The Netherlands). Cell sorting was performed to isolate CD34+/CD38− and CD34+/CD38+ cells from patients and controls, defined as LSCs and HSCs, and L-blasts and control blasts (C-blasts), respectively. Sorting was performed according to the method of Depreter and colleagues [19 (link)], and a detailed description can be found in the Supplemental Material and Methods. Sorted cells were collected in RPMI supplemented with 50% FCS, and a post-sort purity of >90% was reached. Antibodies used for sorting are shown in Table S1 . Sorted cells were spun down (10 min, 3000 rpm, 4 °C) and resuspended in 700 μL of TRIzol for RNA extraction.
3
Isolation and Activation of Primary PBMCs
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Human primary PBMCs were isolated from whole blood of healthy donors or patients, as previously described56 (link). Briefly, mononuclear cells were isolated by Ficoll density gradient (Axis Shield—1114547-1) centrifugation. Cells were activated using PMA (Sigma–Aldrich P1585) (2.5 ng/ml) and Ionomycin (Merck Millipore—407950) (1 µg/ml) or left inactivated in the presence or absence of SMC#13 for 24 h.
4
Isolation and Purification of cDC2 Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy volunteers (Sanquin) and purified via centrifugation over a Ficoll density gradient (Axis-Shield) in SepMate tubes (Stemcell technologies). cDC2s (CD1c+) cells were purified with magnetic cell sorting (MACS) from healthy donor PBMCs using the CD1c (BDCA1) DC isolation kit (Miltenyi Biotec). To obtain a highly purified CD1c+CD14− population, a pre-depletion step of monocytes using CD14-MACS microbeads (Miltenyi Biotec) was included in the original manufacturer’s protocol. DC purity was assessed by staining with primary directly labelled antibodies: anti-CD1c, anti-CD14 and anti-CD20 Abs (Supplementary Table 1 ). Purity levels higher than 96% were achieved, determined by flow cytometry (Supplementary Fig. 6a ). For live imaging experiments, cDC2s were pre-labelled with the membrane dye PKH26 (Sigma-Aldrich) according to manufacturers’ protocols and resuspended in X-VIVO-15 medium (Lonza), supplemented with 2% human serum (HS, Sanquin). Autologous or allogeneic CD3+ T cells used in T-cell co-culture experiments were isolated using the Pan T-cell isolation kit (Miltenyi Biotec) following the manufacturer’s instructions. Peripheral blood monocytes were isolated using CD14-MACS microbeads (Miltenyi Biotec) according manufacturer’s instructions.
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