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Percoll density gradient medium

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Percoll density gradient medium is a laboratory product used to separate and purify cells, organelles, and other biological particles based on their density. It consists of colloidal silica particles coated with polyvinylpyrrolidone, which creates a stable density gradient upon centrifugation. The medium can be used to isolate a variety of cell types, including stem cells, immune cells, and subcellular organelles.

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Lab products found in correlation

Interleukin 6 (il 6), by Thermo Fisher Scientific (17 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (17 mentions) Stemmacs ips brew xf medium, by Miltenyi Biotec (17 mentions) Cytotune ipsc 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (16 mentions) Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (16 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (15 mentions) Stempro 34 sfm medium, by Thermo Fisher Scientific (12 mentions) Stempro 34 medium, by Thermo Fisher Scientific (8 mentions) Matrigel coated plate, by Corning (3 mentions) Scf supplement, by Thermo Fisher Scientific (2 mentions) Cytotune ipsc sendai reprogramming kit, by Thermo Fisher Scientific (1 mentions) Flow cytometry, by Cytek (1 mentions) 40 μm nylon cell strainer, by BD (1 mentions) Nsg mice, by Jackson ImmunoResearch (1 mentions) Tumor dissociation kit, by Miltenyi Biotec (1 mentions) 40 m nylon cell strainer, by BD (1 mentions) Facscalibur, by BD (1 mentions) Cellquest software, by BD (1 mentions)

20 protocols using percoll density gradient medium

1

Efficient PBMC Isolation and iPSC Generation

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PBMCs were isolated from whole blood samples using Percoll density gradient medium (17089109, GE Healthcare). Cells were purified with multiple rounds of DPBS wash (14190144, ThermoFisher Scientific). Cells were cultured in complete PBMC medium composed of StemPro®−34 SFM medium (10639011, ThermoFisher Scientific) containing 100 ng/mL SCF (300–07, Peprotech), 100 ng/mL FLT3 (PHC9414, ThermoFisher Scientific), 20 ng/mL IL-3 (200–3, Peprotech), 20 ng/mL IL-6 (PHC0063, ThermoFisher Scientific), and 20 ng/mL EPO (PHC9631, ThermoFisher Scientific). The medium was refreshed daily until the cell count remained stable for a few days. PBMC transduction was performed using the Sendai virus reprogramming cocktail according to the manufacturer’s instructions (CytoTune™-iPSC 2.0 Sendai Reprogramming Kit, A16517, ThermoFisher Scientific). The transduced cells were resuspended and plated in a Matrigel-coated plate (356231, Corning) and cultured in StemPro™−34 medium (Thermo Fisher). Media was replaced daily until day 7 post transduction. On Day 7, the medium was switched to StemMACS™ iPS-Brew XF medium (130–104–368, Miltenyi Biotec) and was replaced every other day until Day 10–15 post transduction until the colonies appeared. Selected colonies were expanded and frozen down until experimental usage.
Yildirim Z., Kojic A., Yan C.D., Wu M.A., Vagelos R, & Wu J.C. (2022). Generation of two induced pluripotent stem cell lines from dilated cardiomyopathy patients caused by heterozygous mutations in the HCN4 gene. Stem cell research, 65, 102951.
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2

Efficient Generation of iPSCs from PBMCs

Cited 1 time
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PBMCs were isolated from blood by Percoll density gradient medium (GE Healthcare #17089109), purified using DPBS, and plated in a 24-well plate as previously described (Jahng et al., 2021 (link)). PBMCs (~1-2 × 106) were cultured in 1 mL of complete Stem-Pro™−34 medium (Thermo Fisher #14190144) combined with its specific supplements: 100 ng/ mL FLT3 (Thermo Fisher #PHC9414), 20 ng/ mL IL-6 (Thermo Fisher #PHC0063), 20 ng/ mL EPO (Thermo Fisher #PHC9631), 20 ng/ mL IL-3 (Peprotech #200-3), and 100 ng/ mL SCF (Peprotech #300-07). iPSC reprogramming was performed using the Sendai virus reprogramming cocktail based on instructions of the CytoTune®-iPSC Sendai Reprogramming Kit (Thermo Fisher #A16517). The transduced cells were replated in 12-well Matrigel-coated plates. Cells were cultured in Stem-Pro™−34 medium, and the medium was replaced every two days until day 7. On day 7, the medium was switched to supplemented StemMACS™ iPS-Brew XF medium (Miltenyi Biotec #130-104-368). Fresh StemMACS™ iPS-Brew XF medium was changed every two days until days 10-15 when colonies appeared. Then the colonies were prepared for picking and expanded as previously described (Jahng et al., 2021 (link)).
Zhang H., Jahng J.W., Liu Y., Chase A.J., Perez M.V, & Wu J.C. (2021). Generation of three induced pluripotent stem cell lines (SCVIi014-A, SCVIi015-A, and SCVIi016-A) from patients with LQT1 caused by heterozygous mutations in the KCNQ1 gene. Stem cell research, 55, 102492.
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3

Efficient PBMC Reprogramming and iPSC Generation

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PBMCs were isolated from blood using Percoll density gradient medium (#17089109, GE Healthcare), purified using DPBS, and plated in a 24-well plate as previously described (Belbachir et al., 2021 (link)). Cells were cultured in StemPro®-34 SFM medium (#10639011, Thermo Fisher Scientific) and nourished with specific supplements: SCF (100 ng/mL, #300–07, Peprotech), FLT3 (100 ng/mL, #PHC9414, Thermo Fisher Scientific), IL-3 (20 ng/mL, #200–3, Peprotech), IL-6 (20 ng/mL, #PHC0063, ThermoFisher Scientific), and EPO (20 ng/mL, #PHC9631, Thermo Fisher Scientific). PBMCs were reprogramed according to the instructions provided with CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (#A16517, Thermo Fisher Scientific). Transduced PBMCs were resuspended and plated on a Matrigel-coated plate. Cells were cultured in StemPro™-34 medium (Thermo Fisher Scientific). On day 7, the medium was redirected to StemMACS™ iPS-Brew XF medium (#130–104–368, Miltenyi Biotec), and cells were maintained until days 10–15 post-transduction. Cell colonies were picked, and clones expanded as previously described (Belbachir et al., 2021 (link)).
Manhas A., Jahng J.W., Vera C.D., Shenoy S.P., Knowles J.W, & Wu J.C. (2022). Generation of two iPSC lines from hypertrophic cardiomyopathy patients carrying MYBPC3 and PRKAG2 variants. Stem cell research, 61, 102774.
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4

Efficient PBMC Reprogramming and iPSC Generation

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PBMCs were isolated from blood using Percoll density gradient medium (#17089109, GE Healthcare), purified using DPBS, and plated in a 24-well plate as previously described (Belbachir et al., 2021 (link)). Cells were cultured in StemPro®-34 SFM medium (#10639011, Thermo Fisher Scientific) and nourished with specific supplements: SCF (100 ng/mL, #300–07, Peprotech), FLT3 (100 ng/mL, #PHC9414, Thermo Fisher Scientific), IL-3 (20 ng/mL, #200–3, Peprotech), IL-6 (20 ng/mL, #PHC0063, ThermoFisher Scientific), and EPO (20 ng/mL, #PHC9631, Thermo Fisher Scientific). PBMCs were reprogramed according to the instructions provided with CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (#A16517, Thermo Fisher Scientific). Transduced PBMCs were resuspended and plated on a Matrigel-coated plate. Cells were cultured in StemPro™-34 medium (Thermo Fisher Scientific). On day 7, the medium was redirected to StemMACS™ iPS-Brew XF medium (#130–104–368, Miltenyi Biotec), and cells were maintained until days 10–15 post-transduction. Cell colonies were picked, and clones expanded as previously described (Belbachir et al., 2021 (link)).
Manhas A., Jahng J.W., Vera C.D., Shenoy S.P., Knowles J.W, & Wu J.C. (2022). Generation of two iPSC lines from hypertrophic cardiomyopathy patients carrying MYBPC3 and PRKAG2 variants. Stem cell research, 61, 102774.
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5

Efficient Generation of iPSCs from PBMCs

Cited 1 time
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Isolation of PBMCs was carried out using Percoll density gradient medium (GE Healthcare #17089109) and purification by washing with DPBS (ThermoFisher Scientific #14190144). The PBMCs were cultured in 24-well plates with StemPro®−34 SFM medium (ThermoFisher Scientific #10639011) using 100 ng/mL SCF supplement (Peprotech #300–07), 100 ng/mL FLT3 (ThermoFisher Scientific #PHC9414), 20 ng/mL IL-3 (Peprotech #200–3), 20 ng/mL IL-6 (ThermoFisher Scientific #PHC0063), and 20 ng/mL EPO (ThermoFisher Scientific #PHC9631). Reprogramming into iPSCs using the CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific #A16517) was done following the manufacturer’s instructions. The cells were resuspended and plated in a Matrigel-coated plate and cultured in StemPro™-34 medium. On day 7 post-transduction, the medium was switched to StemMACS™ iPS-Brew XF medium (Miltenyi Biotec #130–104–368) until day 10–15. At this stage, colonies appeared that were used for clone picking. The selected colonies were then expanded and frozen down as previously described (Manhas et al., 2022 (link)).
Ringwald M., Eppig J.T., Begley D.A., Corradi J.P., McCright I.J., Hayamizu T.F., Hill D.P., Kadin J.A, & Richardson J.E. (2001). The Mouse Gene Expression Database (GXD). Nucleic Acids Research, 29(1), 98-101.
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6

Generation of iPSCs from PBMCs

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PBMCs were isolated from patient blood using Percoll density gradient medium (GE Healthcare) and purified with several DPBS washes (Thermo Fisher Scientific #14190144). PBMCs were cultured in StemPro®−34 SFM medium (Thermo Fisher Scientific) supplemented with 100 ng/mL SCF (Peprotech), 100 ng/mL FLT3 (Thermo Fisher Scientific #PHC9414), 20 ng/mL IL-3 (Peprotech), 20 ng/mL IL-6 (Thermo Fisher), and 20 ng/mL EPO (Thermo Fisher Scientific #PHC9631). iPSC Sendai virus reprogramming was performed using the CytoTune™-iPSC2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. The transduced cells were resuspended and plated onto a Matrigel-coated plate where they were cultured in StemPro™-34 medium. On day 7 post-transduction, the medium was switched to StemMACS™ iPS-Brew XF medium (Miltenyi Biotec) until day 10–15 post-transduction when colonies appeared ready for clone picking. Selected colonies were expanded over multiple passages and frozen down until experimental usage.
Lee C., Cho S., Lai C., Shenoy S., Vagelos R, & Wu J.C. (2021). Generation of three iPSC lines from dilated cardiomyopathy patients carrying a pathogenic LMNA variant. Stem cell research, 59, 102638.
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7

Generation of iPSCs from PBMCs

Cited 1 time
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PBMCs were isolated from blood by Percoll density gradient medium (GE Healthcare #17089109), washed with DPBS, and plated in a 24-well plate. The culture medium for the PBMCs consisted of Stem-Pro™−34 medium (Thermo Fisher #14190144) supplemented with 100 ng/mL FLT3 (Thermo Fisher #PHC9414), 20 ng/mL IL-6 (Thermo Fisher #PHC0063), 20 ng/mL EPO (Thermo Fisher #PHC9631), 20 ng/mL IL-3 (Peprotech #200–3), and 100 ng/mL SCF (Peprotech #300–07). To reprogram PBMCs into iPSCs, we used the Sendai virus reprogramming cocktail according to the CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific #A16517). The transduced cells were resuspended and plated in a Matrigel-coated plate using the PBMC culture media. On day-7 after transduction, the medium was switched to StemMACS™ iPS-Brew XF medium (Miltenyi Biotec #130–104–368) until day 10–15 post-transduction, when colonies appeared. Colonies were picked and expanded (Manhas et al., 2022 (link)).
Vera C.D., Manhas A., Shenoy S.P., Wheeler M.T., Sallam K, & Wu J.C. (2022). Generation of two induced pluripotent stem cell lines carrying the phospholamban R14del mutation for modeling ARVD/C. Stem cell research, 63, 102834.
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8

iPSC Reprogramming from PBMCs

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PBMCs were isolated from whole blood with Percoll density gradient medium (GE Healthcare #17089109) and purified by washing with DPBS multiple times (ThermoFisher Scientific #14190144). The PBMCs were cultured in StemPro®-34 SFM medium (ThermoFisher Scientific #10639011) supplemented with 100 ng/mL SCF (Peprotech #300–07), 100 ng/mL FLT3 (ThermoFisher Scientific #PHC9414), 20 ng/mL IL-3 (Peprotech #200–3), 20 ng/mL IL-6 (ThermoFisher Scientific #PHC0063), and 20 ng/mL EPO (ThermoFisher Scientific #PHC9631). iPSC reprograming was performed using the CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific #A16517) according to the instructions. The transduced cells were resuspended and plated in a Matrigel-coated plate. Cells were cultured in StemPro™-34 medium. On day 7 post-transduction, the medium was switched to StemMACS™ iPS-Brew XF medium (Miltenyi Biotec #130–104-368) until day 10–15 post-transduction when colonies appeared and were ready for clone picking. The selected colonies were expanded and frozen down until experimental usage.
Sanchez Y., Lovell M., Marin M.C., Wong P.E., Wolf-Ledbetter M.E., McDonnell T.J, & Killary A.M. (1996). Tumor suppression and apoptosis of human prostate carcinoma mediated by a genetic locus within human chromosome 10pter-q11.. Proceedings of the National Academy of Sciences of the United States of America, 93(6), 2551-2556.
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9

Reprogramming PBMCs to iPSCs

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PBMCs were isolated from patient blood using Percoll density gradient medium (GE Healthcare) and purified with several DPBS washes (Thermo Fisher Scientific #14190144). PBMCs were cultured in StemPro®−34 SFM medium (Thermo Fisher Scientific) supplemented with 100 ng/mL SCF (Peprotech), 100 ng/mL FLT3 (Thermo Fisher Scientific #PHC9414), 20 ng/mL IL-3 (Peprotech), 20 ng/mL IL-6 (Thermo Fisher), and 20 ng/mL EPO (Thermo Fisher Scientific #PHC9631). iPSC reprogramming was performed using the CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. The transduced cells were resuspended and plated onto a Matrigel-coated plate where they were cultured in StemPro™−34 medium. On day 7, the medium was switched to StemMACS™ iPS-Brew XF medium (Miltenyi Biotec) until day 10–15 post-transduction when colonies appeared ready for clone picking. Selected colonies were expanded over multiple passages and frozen down until experimental usage.
Cho S., Lee C., Lai C., Zhuge Y., Haddad F., Fowler M., Sallam K, & Wu J.C. (2022). Heterozygous LMNA mutation-carrying iPSC lines from three cardiac laminopathy patients. Stem cell research, 59, 102657.
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10

Generating Human iPSCs from PBMCs

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The methodology was described in the previous study (Zeng et al., 2023 (link)). Briefly, we started by isolating PBMCs from whole blood using Percoll density gradient medium (GE Healthcare #17089109). Then, they underwent multiple washes with DPBS (ThermoFisher Scientific #14190144). Following the isolation, the PBMCs were cultured in StemPro®−34 SFM medium (ThermoFisher Scientific #10639011), which was supplemented with 100 ng/mL SCF (Peprotech #300–07), 100 ng/mL FLT3 (ThermoFisher Scientific #PHC9414), 20 ng/mL IL-3 (Peprotech #200–3), 20 ng/mL IL-6 (ThermoFisher Scientific #PHC0063), and 20 ng/mL EPO (ThermoFisher Scientific #PHC9631). To initiate iPSC reprogramming, we followed the instructions by CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific #A16517). The transduced cells were then resuspended and placed onto Matrigel-coated plates (coated at 1:500 dilution, Corning #356231). StemPro™−34 medium was used to culture these cells. The culture media was exchanged for StemMACS™ iPS-Brew XF medium (Miltenyi Biotec #130–104-368) at day 7 post-transduction, and maintained until colonies appeared, typically occurring on day 10–15 post-transduction. Following this, the colonies were picked and expanded, and then kept frozen down until experimental usage.
Liu W., Zeng W., Kong X., Htet M., Yu R., Wheeler M., Day J.W, & Wu J.C. (2023). Generation of two induced pluripotent stem cell lines from Duchenne muscular dystrophy patients. Stem cell research, 72, 103207.
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