Isolation of Monocytes with High Purity and Yield from Peripheral Blood Mononuclear Cells by Flotation Density Gradient Centrifugation

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15+ cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15+ cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

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Separation and Optimisation of a Sucrose Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells (PBMC)

Asian Journal of Research in Biochemistry ◽

10.9734/ajrb/2018/v3i229820 ◽

2018 ◽

pp. 1-4

Author(s):

Sudeep Nagaraj ◽

Shubha Nivargi ◽

Leelavathy Nanjappa ◽

Jagadish Tavarekere Venkataravanappa

Keyword(s):

Density Gradient ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Mononuclear Cells ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

One Step

One step centrifugation procedure used commonly for separation of blood cells is the ficoll gradient centrifugation. In this method, after centrifugation, the peripheral blood mononuclear cells (PBMCs) are located on the top of the separation fluid, whereas other blood cells erythrocytes and granulocytes sediment to the bottom. In the present study 75% of lymphocyte suspension could be separated by using a one-step density gradient centrifugation of sodium heparin blood with Sucrose. Sucrose was diluted into different concentrations using miliQ water (10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%,). 4 mL of diluted blood was layered on 4 mL of each sucrose solution and centrifuged for 45 minutes at 1000 rpm. Clear separation of PBMCs could be observed in solution with 40% sucrose. The separated PBMCs were analysed in haeme analyser which showed 75% lymphocytes, 23% monocytes and 2% of other cells.

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A QUICK AND EASY METHOD FOR THE ISOLATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM WHOLE BLOOD BY DENSITY GRADIENT CENTRIFUGATION USING HISEP LSM MEDIUM.

International Journal of Advanced Research ◽

10.21474/ijar01/1640 ◽

2016 ◽

Vol 4 (9) ◽

pp. 1774-1778 ◽

Cited By ~ 1

Author(s):

Brahmaji Sontyana ◽

◽

Suresh Govatati ◽

RaghavaRao Tamanam ◽

◽

...

Keyword(s):

Density Gradient ◽

Peripheral Blood Mononuclear Cells ◽

Whole Blood ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Easy Method ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

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Impact of SARS-CoV-2 infection on the recovery of peripheral blood mononuclear cells by density gradient

Scientific Reports ◽

10.1038/s41598-021-83950-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Maria D. I. Manunta ◽

Giuseppe Lamorte ◽

Francesca Ferrari ◽

Elena Trombetta ◽

Mario Tirone ◽

...

Keyword(s):

Density Gradient ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Early Phase ◽

Mononuclear Cells ◽

Early Stage ◽

Gradient Centrifugation ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Circulating Lymphocytes

AbstractSARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms, which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4 ± 27.2%, with a median 28.8% and IQR 11.6–56.1, Welch’s t-test early phase COVID-19 versus blood donor healthy controls P < 0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.

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Impact of SARS-CoV-2 infection on the recovery of peripheral blood mononuclear cells by density gradient

10.21203/rs.3.rs-75145/v1 ◽

2020 ◽

Author(s):

Maria D. I. Manunta ◽

Giuseppe Lamorte ◽

Francesca Ferrari ◽

Elena Trombetta ◽

Mario Tirone ◽

...

Keyword(s):

Density Gradient ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Early Phase ◽

Mononuclear Cells ◽

Early Stage ◽

Gradient Centrifugation ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Circulating Lymphocytes

Abstract SARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4±27.2%, with a median 28.8% and IQR 11.6-56.1, Welch’s t-test early phase COVID-19 versus blood donor healthy controls P<0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.

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Isolation of Peripheral Blood Mononuclear Cells Using Glycerol Density Gradient

Cell Biochemistry and Biophysics ◽

10.1007/s12013-013-9744-z ◽

2013 ◽

Vol 68 (3) ◽

pp. 583-585 ◽

Cited By ~ 2

Author(s):

I. A. Aimola ◽

H. M. Inuwa ◽

A. J. Nok ◽

A. I. Mamman ◽

N. Habila ◽

...

Keyword(s):

Density Gradient ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

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Similar dysregulation of lupus-associated miRNAs in peripheral blood mononuclear cells and splenic lymphocytes in MRL/lpr mice

Lupus Science & Medicine ◽

10.1136/lupus-2018-000290 ◽

2018 ◽

Vol 5 (1) ◽

pp. e000290 ◽

Cited By ~ 6

Author(s):

Zhuang Wang ◽

Bettina Heid ◽

Rujuan Dai ◽

Sattar Ansar Ahmed

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Lupus Erythematosus ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Gradient Centrifugation ◽

Splenic Lymphocytes ◽

Murine Lupus ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Mirnas Expression

ObjectiveMicroRNAs (miRNAs) play an important role in the pathogenesis of various autoimmune diseases including systemic lupus erythematosus (SLE; lupus). We have previously reported a common pattern of miRNA dysregulation in splenic lymphocytes from several mouse models of lupus. In this study, we investigated whether there is a similar miRNAs expression dysregulation in peripheral blood mononuclear cells (PBMCs) and splenocytes in a classical murine lupus model, MRL/lpr.MethodPBMCs were isolated from blood samples of control MRL and lupus MRL/lpr mice aged 14–15 weeks by gradient centrifugation with Histopaque 1083 density media. miRNA TaqMan assays were performed to analyse the expression of 10 lupus-associated miRNAs including miR-182-96-183 cluster, miR-146a, miR-148a, miR-21, miR-31, miR-127, miR-155, and miR-411 in MRL and MRL/lpr PBMCs.ResultIn this study, we found that 8 out of 10 examined miRNAs (miR-21, miR-31, miR-127, miR-155, miR-96, miR-182, miR-183 and miR-411) were similarly dysregulated in both PBMCs and splenocytes of MRL/lpr mice when compared with MRL control mice. Only two miRNAs (miR-146a and miR-148a) showed different dysregulation pattern in the PBMCs and splenocytes of MRL/lpr mice. By comparing with the published miRNA data in human lupus, we demonstrated similarity in miRNA dysregulation in murine and human lupus PBMCs.ConclusionThe findings in this study suggest that the miRNA changes observed in PBMCs largely reflect the miRNA dysregulation in cells from the lymphoid organ spleen. Analysis of miRNAs in PBMCs has an advantage over the splenocytes since it allows for monitoring the kinetics of lupus-associated miRNAs expression with peripheral blood cell samples during the development of the disease or after instituting treatment. The similar dysregulation of miRNAs in murine and human lupus PBMCs supports the importance and the feasibility of using murine lupus models to study the pathogenic and therapeutic function of miRNAs in human lupus.

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Purification of viable peripheral blood mononuclear cells for biobanking using a robotized liquid handling workstation

Journal of Translational Medicine ◽

10.1186/s12967-019-2125-7 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 1

Author(s):

Luigi Coppola ◽

Giovanni Smaldone ◽

Alessandra Cianflone ◽

Simona Baselice ◽

Peppino Mirabelli ◽

...

Keyword(s):

Density Gradient ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Ethylenediaminetetraacetic Acid ◽

Peripheral Blood Mononuclear ◽

Liquid Handling ◽

Blood Mononuclear Cells ◽

Set Up ◽

Automated Protocol

Abstract Background The purification of peripheral blood mononuclear cells (PBMCs) by means of density gradient (1.07 g/mL) centrifugation is one of the most commonly used methods in diagnostics and research laboratories as well as in biobanks. Here, we evaluated whether it was possible to set up an automated protocol for PBMC purification using a programmable liquid handling robotized workstation (Tecan, Freedom EVO 150). We selected a population composed of 30 subjects for whom it was possible to dispose of two ethylenediaminetetraacetic acid (EDTA) vacutainer tubes containing unfractionated peripheral blood. The purification of PBMCs was performed in parallel using automated and manual workflows. Results An automated workflow using the Freedom EVO 150 liquid handling workstation was generated for the isolation of PBMCs. This protocol allowed blood dilution in Dulbecco’s phosphate-buffered saline (DPBS), stratification onto the density gradient, and the collection of PBMC rings after centrifugation. The comparison between the automated and manual methods revealed no significant differences after separation in terms of total mononuclear cell enrichment, red blood cell contamination, or leucocyte formula, including the percentage of lymphoid subpopulations as B, T and natural killer (NK) lymphocytes. Conclusions Our results show that it is possible to set up an automated protocol for the isolation of PBMCs using a robotized liquid handling workstation. This automated protocol provided comparable results to the routinely used manual method. This automatic method could be of interest for those working in biobanking or industries involved in diagnostics and therapeutics field, to avoid operator-dependent errors as well as procedures standardization.

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