Gastrocnemius and quadriceps of mice were minced and digested in trypsin (0.05%). The resulting cells were then incubated with CD45 microbeads (Miltenyi Biotech, Paris, France) and passed on a separation column (Miltenyi Biotech). CD45- and CD45+ were then collected following manufacturer's instructions.
Separation column
Manufactured by Miltenyi Biotec
Sourced in France, Germany, United States
Separation columns are laboratory devices used to separate and purify different components from a mixture. They are designed to provide efficient and reliable separation of target molecules, cells, or particles based on their physical or chemical properties.
Automatically generated - may contain errors
Lab products found in correlation
Cd14 magnetic microbeads, by Miltenyi Biotec (2 mentions)
Minimacs separator, by Miltenyi Biotec (2 mentions)
Cd90 thy1.2 microbeads, by Miltenyi Biotec (2 mentions)
Cd45 microbeads, by Miltenyi Biotec (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Chromium single cell instrument, by 10x Genomics (1 mentions)
Dead cell removal microbeads, by Miltenyi Biotec (1 mentions)
Facscalibur, by BD (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
Easysep mouse myeloid derived suppressor cell isolation kit, by STEMCELL (1 mentions)
Anti f4 80 microbeads ultrapure kit, by Miltenyi Biotec (1 mentions)
Cd4 microbeads, by Miltenyi Biotec (1 mentions)
Mouse mdsc isolation kit, by Miltenyi Biotec (1 mentions)
Macs multistand, by Miltenyi Biotec (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
5 bromo 2 deoxy uridine labeling and detection kit 3, by Roche (1 mentions)
Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Recombinant mouse granulocyte macrophage colony stimulating factor gm csf, by R&D Systems (1 mentions)
Fitc or pe conjugated monoclonal antibodies abs, by BD (1 mentions)
Non labeled abs, by BD (1 mentions)
13 protocols using separation column
1
Isolation of Muscle Cell Subsets
2
Single-cell RNA-seq of CLS1/CAFs Coculture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CLS1 cells (5×103 cells/10 cm dish) were cocultured with CAFs (5×105 cells/10 cm dish) for 5 days and then treated with or without digoxin (1 nM) for 72 h. The CLS1/CAFs coculture mixture cells were collected by trypsin. The cells were suspended in 100 μL of Dead Cell Removal MicroBeads (Miltenyi Biotec; 130-090-101) and incubated for 15 min at room temperature. The separation column (Miltenyi Biotec; 130-042-201) was rinsed with 3 mL of separation buffer (PBS containing 0.5% BSA, 2 mM EDTA, pH=7.2). Then, a cell suspension was applied to the column, and the live cells were collected by washing four times with 3 mL of separation buffer. Live single cells were loaded on a Chromium Single Cell Instrument (10× Genomics, Pleasanton, CA), and cDNA amplification and library construction steps were performed according to the manufacturer's instructions. The single-cell libraries were sequenced using the Illumina NextSeq 500 system (parameters: paired-end sequencing with dual indexing, 26 cycles for Read 1, 8 cycles for I7 Index Read and 98 cycles for Read 2).
3
Magnetic Bead-based Separation of HUVECs and BMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cocultured cells were suspended by trypsin digestion, rinsed with PBS twice, centrifuged at 1500 rpm for 5 min, and collected in 1.5 mL centrifuge tubes. After rinsing with PBE buffer (PBS with 0.5% BSA and 2 mM EDTA, pH 7.2), each sample was retained 100 μL liquid, followed by incubation in CD31 antibody-labeled magnetic bead suspension (Miltenyi, Germany) at 4 °C for 30 min in a dark room. The samples were centrifuged at 1000 rpm for 5 min and rinsed with PBE buffer twice. Then 500 μL PBE was added buffer to obtain the cell suspension. The separation column (Miltenyi) was placed in a magnetic field and wet with 500 μL PBE buffer. Then, cell suspension was added to the separation column and eluted with PBE buffer, and negative cells (BMSCs) were collected. After microscopy of eluent containing 1–2 cells, the separation column was removed from the magnetic field, and PBE buffer was added to elute the positive cells (HUVECs). Cells were incubated with antibody anti-CD31 (PE-conjugated mouse IgG1, R&D, USA) according to the manufacturer’s instructions and analyzed on a flow cytometer (BD FACS Calibur, USA).
4
Isolation and Treatment of Lung Fibroblasts and BM Cells
5
Isolation and Purification of CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The collected spleen was placed into a 5 ml EP tube lled with cold phosphate buffer saline (PBS). Then, the spleen was minced and ground with a syringe piston in a 70 µm sieve to produce a single cell suspension. After centrifugation at 1500 rpm for 5 minutes, the isolated splenocytes were resuspended with PBS. The splenocytes suspension was carefully added to the upper layer of an equal amount of lymphocyte separation solution with a pipette, and the mixed solution was centrifuged at 3000 rpm for 20 minutes. Then, the cloudiness ring-shaped lymphocyte in the middle layer was carefully pipetted into a new 15 ml centrifuge tube, and wash three times with PBS. After cell counting, resuspended each 10 7 cells with 80 µl PBS were mixed with 20 µl of CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for incubation for 15 minutes at 4 °C. The CD4 MicroBeads labeled CD4 + T cells were obtained by a separation column (Miltenyi Biotec) in the magnetic eld of a MACS separator. After washing and centrifugation, the nal cell concentration was determined to be 2 × 10 6 cells / ml and then resuspended in RPMI 1640 containing 10% heat-inactivated fetal bovine serum and 1% antibiotic at 37 °C.
6
Isolation and Purification of Monocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The buffy coat received from a healthy donor (Red Cross, Suarlée, Belgium) was used for monocytes isolation. Peripheral blood mononuclear cells (PBMCs) were separated based on the density gradient of Ficoll™ 1.077 g/mL (Lymphosep BioWest, Nuaillé, France) and centrifuged for 20 min at 900× g without deceleration. Three phases were formed after centrifugation: erythrocytes and granulocytes settled at the bottom of the tube and the platelets in the supernatant, while monocytes and lymphocytes formed a ring between the two phases. This ring was collected, washed with HBSS (Hank’s Balanced Salts Solution, Gibco), and centrifuged for 10 min at 450× g with a deceleration of 9. Then, PBMCs were resuspended with FBS/DMSO 10% and counted in order to be stored at a density of 25 million per cryotube.
Then, CD14+ magnetic microbeads (Miltenyi Biotec, Leiden, The Netherlands) and separation columns (Miltenyi Biotec, Leiden, The Netherlands) were used to isolate the CD14+ monocytes with MiniMACS Separator (Miltenyi Biotec, Leiden, The Netherlands) equipment. The primary monocytes obtained were used for the establishment of the coculture.
Then, CD14+ magnetic microbeads (Miltenyi Biotec, Leiden, The Netherlands) and separation columns (Miltenyi Biotec, Leiden, The Netherlands) were used to isolate the CD14+ monocytes with MiniMACS Separator (Miltenyi Biotec, Leiden, The Netherlands) equipment. The primary monocytes obtained were used for the establishment of the coculture.
7
MDSC Subtype Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of CD11b+ granulocyte receptor 1–positive (Gr‐1+) total MDSCs, CD11b+ lymphocyte antigen 6 complex locus G‐negative (Ly6G‐) lymphocyte antigen 6 complex locus C‐high (Ly6Chi) Mo‐MDSCs, and CD11b+Ly6ClowLy6Ghi Gr‐MDSCs was performed by using mouse MDSC isolation kit (130‐094‐538; Miltenyi Biotech, Auburn, CA) according to the manufacturer’s instruction. Briefly, after Fc receptor blockade, cells were stained with biotin‐conjugated Gr‐1 or Ly6G antibody and further labeled with antibiotin microbeads. Labeled cells were passed through the separation columns (Miltenyi Biotech) for magnetic cell separation. Retained cells were analyzed for the CD11b+Gr‐1+/CD11b+Ly6G–Ly6Chi/CD11b+Ly6ClowLy6Ghi population to assess MDSC purity (>90%) by flow cytometry.
8
Spleen-Derived T-Cell Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One day after injury, mice were sacrificed and spleens were collected. To prepare single cell suspensions, spleens were gently crushed in HBSS solution (Fisher Scientific, Pittsburgh, PA) supplemented with 10 mMol/L HEPES, 50 µg/ml gentamicin and 100 U/ml penicillin with 100 µg/ml streptomycin [12] (link), [13] (link). Cell suspensions were centrifuged at 290 g for 15 min at 10°C. Supernatants were discarded and cells were reconstituted in 9 ml of sterile-distilled H2O following by 1 ml of 10× phosphate-buffered saline (PBS) to lyse red blood cells. 106–107 total cells were resuspended in 90 µl of staining buffer (PBS containing 0.5% BSA and 2 mMol/L EDTA) and incubated with 10 µl of CD90 (Thy1.2) MicroBeads (MiltenyiBiotec, Auburn, CA) for 15 min at 4°C. The cells were washed with staining buffer and run through separation columns (Miltenyi Biotec) in a magnetic field. Purified T cells were obtained by flushing out magnetically labeled cells from the separation columns [13] (link).
9
Isolation of CD14+ Monocytes from Buffy Coat
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Buffy coat blood from a healthy anonymous donor (Croix Rouge, Service du sang, Suarlée, Belgium) was used for monocytes isolation. The formed elements of the blood were separated by a high-density gradient of Ficoll 1.077 g/mL (Lymphosep, VWR L0560-100, Nuaillé, France). Erythrocytes and granulocytes settled at the bottom of the tube while lymphocytes and monocytes remained at the sample–separation medium interface and the platelets were in the supernatant. The cell ring containing the Peripheral Blood Mononuclear Cells (PBMC) was collected. Then, the CD14+ monocytes were isolated from PBMC using CD14+ magnetic microbeads (Miltenyi Biotec, Leiden, The Netherlands). Separation columns (Miltenyi Biotec, Leiden, The Netherlands) were used to isolate the CD14+ monocytes with MACS MultiStand (Miltenyi Biotec, Leiden, The Netherlands) and MiniMACS Separator (Miltenyi Biotec, Leiden, The Netherlands) equipment. Monocytes from PBMC were then cultured in RPMI 1640 like THP1.
10
Murine Dendritic Cell Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (rmIL)-4 were purchased from R&D Systems (Minneapolis, MN, USA), Dextran-FITC (40,000 Da), propidium iodide (PI), ovalbumin (OVA), and mitomycin C (MMC) were purchased from Sigma-Aldrich (Steinheim, Germany), and carboxyfluorescein succinimidyl ester (CFSE), lipopolysaccharide (LPS), and Pam3CSK4 (Pam3) were purchased from Invitrogen (Carlsbad, CA, USA). The following FITC- or PE- conjugated monoclonal antibodies (Abs) and non-labeled Abs were purchased from BD Biosciences (San Jose, CA, USA) : FITC-annexin V, CD16/32 (2.4G2), CD11c (HL3), CD40 (HM40–3), CD80 (16–10A1), CD86 (GL1), H-2Kb (AF6-88.5), I-A[b] (AF6–120.1), and CCR7 (4B12). Cytokine ELISA primary and secondary -antibodies specific for murine IL-1β, IL-6, IL-12p70, IFN-γ, IL-4 and TNF-α were purchased from BD Biosciences (San Jose, CA, USA), and the OT-1 peptide (OVA257-264) was purchased from Invivogen (San Diego, CA, USA). CD4+ T cell isolation kit II, CD11c Isolation Kit II and Separation Columns were purchased from MACS Miltenyi Biotec (Auburn, USA). 5-Bromo-2′-Deoxy-Uridine Labeling and Detection Kit III were purchased from Roche (Salt Lake City, UT, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!