Blood samples were collected in EDTA+ tubes. PBMCs were isolated from whole blood by density gradient centrifugation with Ficoll-sodium diatrizoate (Lymfoprep, Axis-Shield PoC AS, Oslo, Norway) (400 × g, 30 min, 20 °C, without break). As such, PBMCs were separated from the red blood cells and neutrophils according to their density. The top layer contained both PBMCs and plasma and after collecting the PBMCs, they were washed twice with Dulbecco’s phosphate buffered saline (DPBS without Ca2+ and Mg2+; Lonza, Bazel, Switzerland) and counted in a hemocytometer.
Lymfoprep
Manufactured by Axis-Shield
Sourced in Norway
Lymfoprep is a density gradient medium used for the isolation of mononuclear cells, such as lymphocytes and monocytes, from whole blood or buffy coat. It is a sterile, pyrogen-free solution that allows the separation of mononuclear cells from other blood components through centrifugation.
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Lab products found in correlation
Dulbecco s phosphate buffered saline dpbs without ca2 and mg2, by Lonza (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Lonza (1 mentions)
Complete lysis m edta free buffer, by Roche (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Greiner (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Alsever s solution, by Merck Group (1 mentions)
4 protocols using lymfoprep
1
PBMC Isolation from Whole Blood
2
Isolation of Polymorphonuclear Leukocytes
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Blood samples were collected in EDTA-coated tubes. To isolate PMNs from whole blood samples, we applied density gradient centrifugation [31 (link)]. The blood samples were diluted with Dulbecco’s phosphate buffered saline (D-PBS; Lonza, Basel, Switzerland), gently layered on Ficoll-sodium diatrizoate (Lymfoprep, Axis-Shield PoC AS, Dundee, United Kingdom) and centrifuged (400× g, 30 min, 20 °C, without break). The bottom layer with PMNs and red blood cells was first diluted with D-PBS and then gently mixed with 6% Dextran (Sigma-Aldrich, St. Louis, MO, USA) in Milli-Q water to induce red blood cell aggregation and sedimentation. After an incubation period of 30 min at 37 °C, PMNs were isolated. We performed two washing steps with D-PBS and lysed residual red blood cells by a hypotonic shock [31 (link)]. After two additional washing steps, PMNs (>95% pure) were ready for use.
3
Optimized PBMC Protein Extraction
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Peripheral blood mononuclear cells (PBMCs) were isolated from 5–10 ml Lithium-Heparin-blood samples using Lymfoprep (Axis Shield PoC AS, Oslo, Norway) and complete Lysis-M EDTA-free buffer (Roche Diagnostics GmbH, Mannheim Germany). Isolation was performed within 4 hours of sample collection. PBMC counts from samples ranged from 1.26x107 to 3.3x107 cells.
We tested three protein extraction buffers using PBMCs that were pelleted and re-suspended in 100 μL of each buffer. Comparison between complete Lysis-M EDTA-free buffer, RIPA buffer and ENZO lysis buffer showed comparable inter-well variability (complete Lysis-M EDTA-free buffer: mean 6.0% CV; RIPA buffer: 5.5% CV; ENZO lysis buffer mean 5.9% CV). Mean inter-plate variability was 35% (range 8.7–90%). We used complete Lysis-M EDTA-free buffer (Roche Diagnostics GmbH, Mannheim Germany) for all samples. Lysates were stored at -80°C in aliquots of 100–200 μl.
We tested three protein extraction buffers using PBMCs that were pelleted and re-suspended in 100 μL of each buffer. Comparison between complete Lysis-M EDTA-free buffer, RIPA buffer and ENZO lysis buffer showed comparable inter-well variability (complete Lysis-M EDTA-free buffer: mean 6.0% CV; RIPA buffer: 5.5% CV; ENZO lysis buffer mean 5.9% CV). Mean inter-plate variability was 35% (range 8.7–90%). We used complete Lysis-M EDTA-free buffer (Roche Diagnostics GmbH, Mannheim Germany) for all samples. Lysates were stored at -80°C in aliquots of 100–200 μl.
4
PBMC Isolation from EDTA-Blood
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The protocol was modified from a previously developed protocol (Goddeeris et al., 1986) . Fifteen milliliters of EDTA-blood was diluted into an equal volume of Alsever's solution (Sigma-Aldrich, Diegem, Belgium) and layered over 20 mL of Lymfoprep (specific gravity = 1.077; Axis-Shield, Oslo, Norway) in 50-mL conical tubes (BD Falcon, Becton Dickinson, Franklin Lakes, NJ) and centrifuged (30 min, 900 × g, 18°C). Cells at the interface were aspirated, transferred to new 50-mL conical tubes and washed with ice-cold Alsever's solution, containing 1% fetal bovine serum (Greiner Bio One, Wemmel, Belgium; 10 min at 450 × g followed by 10 min at 300 × g, 4°C). The resulting cell pellets were resuspended in 1 mL of RPMI 1640 medium (Sigma-Aldrich), containing 10% FBS, 2 mM l-glutamine (Gibco, Life Technologies, Carlsbad, CA), 50 μg/mL of gentamicin (Sigma-Aldrich), and 5 × 10 -5 M 2-mercaptoethanol (Merck, Darmstadt, Germany; hereafter named culture medium). The isolated PBMC were counted in a Neubauer counting chamber using eosin-nigrosin.
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