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Lymphocyte separation solution

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Lymphocyte separation solution is a sterile, isotonic, and density-gradient medium used for the isolation and purification of lymphocytes from whole blood or other cell suspensions. It facilitates the separation of mononuclear cells, including lymphocytes, from other blood components such as erythrocytes and granulocytes.

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Lab products found in correlation

Anti pd 1, by Proteintech (1 mentions) Anti cd8a, by Thermo Fisher Scientific (1 mentions) Anti cd4, by Thermo Fisher Scientific (1 mentions) Anti foxp3, by Thermo Fisher Scientific (1 mentions) Ls column, by Miltenyi Biotec (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dmem low glucose medium, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)

7 protocols using lymphocyte separation solution

1

PBMNC Isolation and Culture Protocol

Cited 2 times
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Fifteen milliliters (ml) of peripheral blood were collected by venous puncture of superficial vein at forearm. PBMNCs were isolated by density gradient centrifugation using Lymphocyte Separation Solution (Sigma-Aldrich Corporation, St. Louis, MO, USA). PBMNCs at a concentration at 2 × 106 cells/2 ml were cultured either in QQ culture media [11 (link)] or in standard culture media [11 (link)] The cells were cultured in a 6-well Primaria dish (BD Biosciences, San Jose, CA, USA) for 7 days [11 (link), 13 (link)].
Chruewkamlow N., Pruekprasert K., Phutthakunphithak P., Acharayothin O., Prapassaro T., Hongku K., Hahtapornsawan S., Puangpunngam N., Chinsakchai K., Wongwanit C., Ruangsetakit C, & Sermsathanasawadi N. (2021). Novel culture media enhances mononuclear cells from patients with chronic limb-threatening ischemia to increase vasculogenesis and anti-inflammatory effect. Stem Cell Research & Therapy, 12, 520.
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2

RPPA Technique for Protein Analysis

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The methodology and validation of the RPPA technique have been described elsewhere. [17 (link)–21 (link)] Briefly, fresh samples were obtained from patients at local sites and were shipped to the central processing lab at Baylor College of Medicine by overnight courier. Mononuclear cells were isolated from peripheral blood by centrifugation using lymphocyte separation solution (Sigma) and enriched for leukemic cells by CD3/CD19 depletion (Miltenyi Biotech, Cologne, Germany). Protein preparations were normalized to a concentration of 1×107 cells/mL and printed in five serial dilutions onto slides along with normalization and expression controls. Slides were probed with 301 antibodies listed in Supplementary Table 1, including a primary validated antibody against total RelA (Cell Signaling, Danvers, MA, Cat. #3034) and RelA-pSer536 (Cell Signaling, Cat. #3033). Five antibodies were excluded for different reasons yielding a final of 296 antibodies used for analysis. [11 ] Stained slides were analyzed using Microvigene® Software (version 3.0, Vigene Tech, Carlisle, MA). SuperCurve algorithms were used to generate a single concentration value from the five serial dilutions. [22 (link)] Loading control [23 (link)] and topographical normalization [24 ] procedures were performed to account for protein concentration and background antibody staining variations on each array.
van Dijk A.D., Hoff F.W., Qiu Y., Gerbing R.B., Gamis A.S., Aplenc R., Kolb E.A., Alonzo T.A., Meshinchi S., Jenkins G., de Bont E.S., Kornblau S.M, & Horton T.M. (2021). Bortezomib is significantly beneficial for de novo pediatric AML patients with low phosphorylation of the NF-κB subunit RelA. Proteomics. Clinical applications, 16(2), e2100072.
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3

Isolation and Characterization of Monocytes

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Venous blood was diluted with Hanks’ solution and slowly added to the tube containing 2 mL lymphocyte separation solution (Sigma, St. Louis, MO, United States) along the tube wall, with the interface kept clear. The sample was then centrifuged at 671 g for 20 min at room temperature. The upper layer of light yellow plasma was carefully discarded, the white layer (monocytes) was carefully aspirated, and 2 mL Hanks’ solution was added, and the sample was mixed and then centrifuged at 377 g for 10 min at room temperature. The supernatant was discarded, and 1 mL Hanks’ solution was added and resuspended, and the sample was temporarily stored at 4 °C. After the joint cavity effusion fluid was centrifuged, the supernatant was aspirated and stored at -80 °C for ELISA. The pellet was added to 1 mL Hanks’ solution and resuspended, and macrophages were analyzed as described above.
Liu S., Song L.P., Li R.B., Feng L.H, & Zhu H. (2021). Iguratimod promotes transformation of mononuclear macrophages in elderly patients with rheumatoid arthritis by nuclear factor-κB pathway. World Journal of Clinical Cases, 9(10), 2181-2191.
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4

Isolation and characterization of T cell subsets

Cited 1 time
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Spleens of recipient rats were collected, grounded, and filtered to obtain cell suspension, which was centrifuged at 1600 rpm at 4°C for 5 mins. The precipitation was resuspended by pre-cooled phosphate buffer solution. Double volume of lymphocyte separation solution purchased from Sigma-Aldrich was added and centrifuged at 2000 RPM for 25 min to obtain cell suspension. CD4+ T cells and CD8+ T cells were isolated by LS column (Miltenyi Biotec, Germany, #130-042-401) with the method of antibiotic microbeads (Miltenyi Biotec, Germany, # 130-090-319, # 130-090-318) as previously described (37 (link), 38 (link)). Sorted cells were incubated directly with diluted fluorochrome-conjugated monoclonal antibodies as shown below: anti-CD4 (Invitrogen, #11-0040-82, FITC), anti-CD8a (Invitrogen, #11-0084-82, FITC), anti-PD-1 (Proteintech, #65211, Coralite647) and anti-Foxp3 (eBioscience, #12-5773-82, PE).
Cao W., Lu J., Li S., Song F, & Xu J. (2022). Transcriptomic analysis of graft liver provides insight into the immune response of rat liver transplantation. Frontiers in Immunology, 13, 947437.
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5

Time-course Analysis of ConA-induced AIH

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Blood samples (~200 µl) were obtained via the tail vein at 6, 12 and 18 h after ConA-induced AIH. At 24 h, the mice were sacrificed via cervical dislocation and ~1 ml blood samples were harvested. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphocyte Separation Solution (Sigma-Aldrich; Merck Millipore) and stored at −80°C.
Qian J., Meng Z., Guan J., Zhang Z, & Wang Y. (2017). Expression and roles of TIPE2 in autoimmune hepatitis. Experimental and Therapeutic Medicine, 13(3), 942-946.
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6

Isolation and Cultivation of Bone Marrow-Derived Cells

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Two millilitres of fresh bone marrow was collected from healthy volunteers who signed the written informed consent (following the Declaration of Helsinki). Immediately after collection, samples were diluted with 2 ml phosphate‐buffered saline (PBS). The mixture was then placed on the fluid level of lymphocyte separation solution (2 ml, Sigma) and centrifuged at 1000 rpm for 30 min. The liquid was divided into four layers. The second layer (white transparent layer) was aspirated, placed in a cell culture bottle with low‐glucose DMEM medium (Gibco) containing 10% foetal bovine serum (Gibco) and 1% Penicillin‐Streptomycin (Gibco) and cultured in an incubator at 37°C and 5% CO2. After 3 days, 4 ml of low‐sugar complete medium was added. Cells were passaged after reaching 80%–90% confluence; the medium was changed every 5 days. After two to three passages, cells were incubated in serum‐free low‐sugar DMEM for 24–48 h (depending on cell density). Consequently, the medium without cells was centrifuged at 3000 rcf for 20 min, and the supernatant was carefully aspirated into a centrifuge tube and frozen at −80°C.
Lu Y., Yang L., Chen X., Liu J., Nie A, & Chen X. (2021). Bone marrow mesenchymal stem cell‐derived exosomes improve renal fibrosis by reducing the polarisation of M1 and M2 macrophages through the activation of EP2 receptors. IET Nanobiotechnology, 16(1), 14-24.
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7

Peripheral Blood Mononuclear Cell Isolation

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Anticoagulated peripheral blood (5 mL) was incubated with lymphocyte separation solution (Sigma, 10,771), and the mononuclear cells were separated by density gradient centrifugation [27 (link)]. Finally, the cells were frozen in liquid nitrogen until further use.
Li Y., Deng Y, & He J. (2021). Monocyte-related gene biomarkers for latent and active tuberculosis. Bioengineered, 12(2), 10799-10811.
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