For murine experiments, BM was isolated from naive mice followed by red blood cell lysis. Human healthy donor blood samples were collected after informed consent and all research was approved by the local ethical committee (B.U.N. 143201316382). Peripheral blood mononuclear cells were obtained from whole blood of healthy donors and separated by Ficoll-Hypaque (Nycomed, Lucron Bioproducts, De Pinte, Belgium) gradient centrifugation. CD11b+ cells were positively selected by the use of CD11b MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions.
Cd11b macs beads
Manufactured by Miltenyi Biotec
Sourced in Germany
CD11b MACS beads are magnetic microbeads coated with anti-CD11b antibodies. They are designed for the isolation and enrichment of CD11b-positive cells from various sample types using magnetic separation techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll hypaque, by Takeda Pharmaceuticals (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Rnaeasy micro kit, by Qiagen (1 mentions)
Cell strainer, by BD (1 mentions)
Papain, by Merck Group (1 mentions)
Rnaeasy plus mini kit, by Qiagen (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Polyethylene tube, by BD (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Optiprep, by Merck Group (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
L glutamine, by Lonza (1 mentions)
8 protocols using cd11b macs beads
1
Isolation and Purification of Murine and Human Myeloid Cells
2
Isolation and Culture of Gr-1+ Myeloid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes were isolated after red blood cell (RBC) lysis. CD11b+ cells were positively selected using CD11b MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany), and then stained with Gr-1 mAb. Gr-1+ myeloid cell populations were isolated through cell sorting. Purified myeloid cells were cultured in RPMI 1640 medium containing 10% FBS, with the addition of glutamine, sodium pyruvate, nonessential amino acid, and antibiotics.
3
Microglia Isolation for RNA-Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For sorting microglia only for downstream RNA-sequencing, cells were isolated as described previously84 (link). Briefly, barrel cortices dissected as described above were mechanically dissociated using a glass tissue homogenizer in isolation medium (HBSS, 15 mM HEPES, 0.6% glucose, 1 mM EDTA pH 8.0). Cells were filtered and then pelleted at 300 g for 10 minutes at 4°C before being resuspended in 22% Percoll (GE Healthcare) and centrifuged at 900 g for 20 minutes with acceleration set to 4 and deceleration set to 1 in order to remove cellular debris. Pelleted microglia were then resuspended in staining media (PBS, 0.5% BSA, 2 mM EDTA) and incubated with CD11b MACS beads (Miltenyi, 1:50) for 15 minutes at 4°C. The cells were washed with staining buffer, pelleted at 300 g for 5 minutes at 4°C, and reconstituted in 500 uL staining buffer. Microglia were isolated as described in the manual for MACS LS columns and collected in staining buffer without EDTA, pelleted at 300 g for 5 minutes at 4°C , counted on a hemocytometer, and 15,000–20,000 cells were diluted in 30 μL in a BSA-coated plate for 10x sequencing.
4
Murine Myeloma Model with Pargyline and Bortezomib
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C57BL/KalwRij mice were purchased from Envigo Laboratories (Horst, The Netherlands). They were housed and treated following conditions approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (licence No LA1230281, CEP No 20–281-6). On day 0, all mice (n = 9/group) were inoculated (I.V.) with 1 million of eGFP positive 5TGM1 cells dissolved in 200 µl of RPMI-1640 medium. Mice were treated intraperitonealy with 100 mpk pargyline 5x/week, and 0.6 mpk bortezomib intraperitonealy 2x/week starting 14 days after inoculation. After 30 days, vehicle mice reached end-stage and all mice were sacrificed. We isolated BM, followed by red blood cell lysis. Tumor burden in the BM was assessed by eGFP positivity on flow cytometry. For western blot, we further purified the 5TGM1 cells by negative selection (CD11b-) using CD11b MACS beads (Miltenyi Biotec) according to the manufacturer’s instructions.
During the experiment, the general health of all mice was evaluated daily, and body weight was measured 2x/week.
During the experiment, the general health of all mice was evaluated daily, and body weight was measured 2x/week.
5
Curcumin Modulates STAT3 and NF-κB in Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear extracts of MC38 and JHOC‐5 treated with curcumin for 6 h in vitro and tumor‐infiltrating CD11c+ and CD11b+ cells purified from MC38‐implanted C57BL/6 mice using CD11c and CD11b MACS beads (Miltenyi Biotec) were prepared by NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Signosis). STAT3 and NFκB transcription activities in nuclear extracts were measured using a STAT3 and NFκB Filter Plate Assay (Thermo Scientific) in accordance with the manufacturer's instructions.
6
Microglia Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From the same harvest, macrophage precursors (pMacpre) were either lysed directly or differentiated to microglia in monoculture (pMGL) or microglia in co-culture with MNs (co-pMG) for 14 days. pMGL were rinsed with PBS and directly lysed in the dish. For both pMacpre and pMGL, RNA was extracted using an RNAeasy Mini Plus kit (Qiagen) according to the manufacturer’s instructions. Co-cultures were first dissociated by 15 min incubation with papain (P4762, Sigma-Aldrich) diluted in accutase (20 U/mL) and gentle trituration based on a previously published protocol40 (link). The cell suspension was then passed through a cell strainer (70 μm, Falcon) to remove cell clumps. To extract co-pMG, magnetic-activated cell sorting (MACS) was then performed using CD11b-MACS beads (130–093-634, Miltenyi Biotec) according to the manufacturer’s instructions. The panned cell population was lysed for RNA extraction using an RNAeasy Micro kit (Qiagen) according to the manufacturer’s instructions. In addition, RNA from human fetal microglia and blood monocytes from three different healthy genetic backgrounds was re-used from our previous study26 (link).
7
Isolation of Intestinal Mononuclear Phagocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of small intestinal mononuclear phagocytes was performed as previously described64 (link) with modifications. Briefly, small intestine isolated from individual mouse was inverted on a polyethylene tube (BD Biosciences), and washed with calcium- and magnesium-free PBS. Then, the mucus was removed by treating with 1 mM dithiothreitol (Sigma). After wash, the intestine was incubated twice with 30 mM EDTA for 10 min to elute epithelium, followed by incubation with 36 U ml−1 collagenase IV (Sigma) and 500 U ml−1 of DNase I (Thermo Scientific) in PBS for 90 min at 37 °C. The digested tissue was then passed through a 70-μm Cell Strainer (BD Biosciences) and washed with DMEM. Mononuclear phagocytes were collected from an OptiPrep (Sigma) density centrifugation. Macrophages were enriched by incubating single-cell suspension obtained above with CD11b MACS beads according to the manufacturer's instructions (Miltenyi Biotec). Bead-attached cells were separated by positive selection using MACS LS magnetic column and cultured in RPMI-1640 with 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 10% fetal bovine serum.
8
Isolation and Purification of MDSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total BM was isolated from na€ ve and diseased 5T33MM mice followed by red blood cell lysis. Total MDSCs were positively selected by CD11b MACS beads (Miltenyi Biotec) according to the manufacturer's instructions. Granulocytic MDSCs were positively selected by Ly6G MACS beads (Miltenyi Biotec). The Ly6G À fraction was collected and further purified by the use of CD11b microbeads to obtain the monocytic MDSC population (Ly6G À CD11b þ ; purity > 90%). Cells were cultured in RPMI-1640 medium (Lonza) with 10% FCS (Biochrom AG) and supplements (100 U/mL penicillin/streptomycin and 2 mmol/L L-glutamine; Lonza).
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!