Lymphocyte separation solution

Manufactured by Solarbio

Sourced in China

Lymphocyte separation solution is a laboratory reagent used to isolate and purify lymphocytes, a type of white blood cell, from whole blood or other biological samples. It utilizes density gradient centrifugation to separate the different components of the sample, allowing the lymphocytes to be collected for further analysis or experimentation.

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7 protocols using lymphocyte separation solution

Isolation of Spleen and Blood Lymphocytes

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Five chickens were randomly selected from each group 14 d after immunization. Their spleens were removed under aseptic conditions, then rinsed 3 times with sterile PBS and placed in a flat dish. They were cut up, ground down and filtered by a tissue sieve to produce a ground spleen suspension. The suspension was slowly added to the upper layer of the lymphocyte separation solution (Solarbio, Beijing, China) at a ratio of 1:1 and centrifuged for 20 min at 3,000 rpm. After centrifugation, the white misty spleen lymphocytes at the interface were carefully aspirated, rinsed twice with PBS and centrifuged for 15 min at 2,500 rpm (Hou et al., 2010 ; Chimeno Zoth et al., 2012 (link)).
On the 14th d after immunization, 2 mL of blood was taken under sterile conditions and anticoagulated with heparin (Solarbio, Beijing, China), diluted 1:1 with PBS and then carefully added this diluted blood to the upper layer of the lymphocyte separation solution (Solarbio, Beijing, China) at a ratio of 1:1 and centrifuged at 2,500 rpm/min for 20 min. Afterward, the peripheral blood lymphocytes at the middle boundary were harvested, washed twice with PBS and centrifuged at 1,500 rpm for 15 min (Lam and Vasconcelos, 1994 (link); Gu et al., 2020 ).

Wu Y., Li N., Zhang T., Che Y., Duan K., Wang Y., Zhou H., Wan X., Lei H., Nguyễn A.D., De Souza C., Li K., Wu Y., Liu J, & Wang D. (2021). Glycyrrhiza polysaccharides can improve and prolong the response of chickens to the Newcastle disease vaccine. Poultry Science, 101(1), 101549.

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Isolation and Transfection of Tonsil Mononuclear Cells

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Tonsil tissues from the IgAN group and CT group were soaked in PBS for 15 min, fully ground, and filtered through a 200 mesh sieve, then the filtrate was collected. Slowly, an equal volume of lymphocyte separation solution was added (Solarbio Technology Co. Beijing, China) and centrifuged at 1,500 rpm for 20 min to retain the middle mononuclear cell layer, and then inactivated 2% fetal bovine serum (FBS, Gibco, Invitrogen) was added. After washing with PBS three times, the cells were resuspended and centrifuged at 1,500 rpm for 5 min. Finally, the tonsil mononuclear cells (TMCs) were cultured in RPMI-1640 medium with 10% FBS, L-glutamine (2 mmol/L), penicillin (100 μg/ml), streptomycin (100 μg/ml) at 37°C in a humidified atmosphere with 5% CO₂. For miR-630 mimic transfection, TransIT-TKO transfection reagent was used and transfected according to the manufacturer’s instructions. Briefly, 25 nM of miR-630 mimic with FAM-labeled (GenePharma, Shanghai, China), 4 µl of TransIT-TKO transfection reagent and 50 µl of Opti-MEM Reduced Medium (Invitrogen, USA) was incubated for 20 min and then mixed with the TMCs derived from one IgAN patient per well. A miRNA negative control was also transfected at the same time. The TMCs were incubated for 48–72 h at 37°C in 5% CO₂ and used for total RNA and protein extraction.

Liu C., Ye M.Y., Yan W.Z., Peng X.F., He L.Y, & Peng Y.M. (2020). microRNA-630 Regulates Underglycosylated IgA1 Production in the Tonsils by Targeting TLR4 in IgA Nephropathy. Frontiers in Immunology, 11, 563699.

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Isolation and Purification of CD19+ B Cells

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The peripheral blood of patients in the HC and OS groups was collected, lymphocyte separation solution (Solarbio, USA) was added, and single-cell suspension was collected after 30 min at 4°C 900 r/min. A CD19 antibody was used to label B cells. Every 10⁷ cells were resuspended with 90 μl of MACS buffer, added with 10 μl of anti-CD19 microbeads after mixing, mixed well by tapping the bottom of the tube, and incubated at 4°C for 15 min in the dark. 1 ml buffer was added to rinse the cells and centrifuged at 300 g for 10 min, and the supernatant was discarded before adding 1 ml buffer to resuspend the cells. The MidiMACS sorter was used to sort and collect CD19⁺ B cells.

Wang Z., Liang J., Jiang S., Zhao G., Lu J, & Jiang B. (2021). The Effect of miR-138 on the Function of Follicular Helper T Cells and the Differentiation of B Cells in Osteosarcoma. Computational and Mathematical Methods in Medicine, 2021, 2057782.

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Anti-inflammatory Effects of Nicotine

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MG (purity >98%) and carboxyl methyl cellulose sodium (CMC-Na) were obtained from SanHerb Bioscience (Chengdu, Sichuan, People’s Republic of China) and Aladdin (Shanghai, People’s Republic of China), respectively. Lipopolysaccharide (LPS, from the Gram-negative bacterium E. coli055:B5), nicotine (Nic, α7nAChR agonist), and methyllycaconitine (MLA, α7nAChR specific inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were bought from Hyclone (Logan City, UT, USA). Lymphocyte separation solution was purchased from Solarbio (Beijing, People’s Republic of China). Anti-human CD4-APC, IL-17A-FITC, CD25-FITC, and CD127-PE-CY7 antibodies were obtained from BioLegend (San Diego, CA, USA). ELISA kits (rat TNF-α, IL-1β; human TNF-α, IL-1β, IL-10) were supplied by Boster Biotech (Wuhan, People’s Republic of China). Anti-rat p65, p-p65, and α7nAChR antibodies were products of Cell Signaling Technology (Boston, MA, USA). Biotin-conjugated secondary antibodies were purchased from Beyotime Biotech (Nantong, Jiangsu, People’s Republic of China).

Yin Q., Wu Y.J., Pan S., Wang D.D., Tao M.Q., Pei W.Y, & Zuo J. (2020). Activation of Cholinergic Anti-Inflammatory Pathway in Peripheral Immune Cells Involved in Therapeutic Actions of α-Mangostin on Collagen-Induced Arthritis in Rats. Drug Design, Development and Therapy, 14, 1983-1993.

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Isolation of BM-Derived Mononuclear Cells

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Fresh heparinized BM samples were diluted with phosphate-buffered saline (PBS) in a 1:1 proportion. The same volume of lymphocyte separation solution (Solarbio, China) as the mixture was added to the centrifuge tube. The diluted BM samples were carefully layered on lymphocyte separation solution and centrifuged at 400g for 20min. A clean pipette was used to transfer the lymphocyte layer to a clean centrifuge tube and then further washed with PBS. The supernatant was discarded. Red blood cell (RBC) lysis solution (Solarbio, China) was added into the tube and incubated for 8 min away from light, centrifuged, and washed with PBS again. We obtained BMMNCs for follow-up experiments.

Zhang Y., Hao S., Xiao N., Zhang Y., Wang H., Li L., Fu R, & Shao Z. (2022). Ferritin Light Chain: A Candidate Autoantigen in Immuno-Related Pancytopenia. Frontiers in Immunology, 13, 851096.

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Adoptive Transfer of Lymph Nodes in Irradiated Mice

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Axillary and inguinal lymph nodes (LNs) were collected from C57BL/6 donors and then ground into homogenates, washed, centrifuged, and filtered. Their cell number was counted and adjusted to 10⁷/100 μL.
The mice were divided into three groups: a normal control group (NC, n = 10), a TBI group (n = 10), and an AA model group (n = 17). The NC group contained CB6F1 mice. The TBI mice were given 4 Gy TBI using a cesium-137 gamma source. The AA model mice were also preirradiated with 4 Gy TBI using the same cesium-137 gamma source. After 4–6 h, diluted LN cells were injected through the retroorbital sinus into the recipient AA group mice (CB6F1) at 10 × 10⁶ cells/recipient in a 100 μL lymphocyte separation solution (Solarbio, Beijing, China).

Zheng M., Liu B., Shao Y., Hua L., Fu R., Wang H., Wang T., Qi W., Shao Z, & Liu C. (2020). Effects of Shikonin on the Functions of Myeloid Dendritic Cells in a Mouse Model of Severe Aplastic Anemia. Mediators of Inflammation, 2020, 9025705.

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Microglia Regulation by MG in LPS-Induced Inflammation

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MG (purity >98%) was purchased from SanHerb Bioscience (Chengdu, Sichuan, China). Carboxyl methyl cellulose sodium (CMC-Na) was obtained from Aladdin (Shanghai, China). Lipopolysaccharide (LPS, from the Gram-negative bacterium E. coli 055:B5) and methyllycaconitine (MLA, α7nAChR specific inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were bought from Hyclone (Logan City, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (CA, USA). Lymphocyte separation solution was bought from Solarbio (Beijing, China). Anti-rat CD4-PE, IFN-γ-FITC, CD86-FITC and CD11b-PE antibodies were purchased from BioLegend (San Diego, CA, USA). Anti-rat p65, ac-p65, p-p65, α7nAChR and SIRT1 antibodies were the products of Affinity Biosciences (Changzhou, Jiangsu, China). Biotin-conjugated secondary antibodies were supplied by Beyotime Biotech (Nantong, Jiangsu, China). Acetylcholinesterase (AChE) assay kit and Coenzyme I NAD (H) assay kit were supplied by JianCheng Bioengineering Institute (Nanjing, Jiangsu, China).

Chen W.G., Zhang S.S., Pan S., Wang Z.F., Xu J.Y., Sheng X.H., Yin Q, & Wu Y.J. (2022). α-Mangostin Treats Early-Stage Adjuvant-Induced Arthritis of Rat by Regulating the CAP-SIRT1 Pathway in Macrophages. Drug Design, Development and Therapy, 16, 509-520.

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