Mice were transplanted as previously described.21 (link) In brief, recipient B6D2F1 mice were intravenously (i.v.) injected with 5×106 TCD-BM cells form WT B6 donors plus 1×106 T cells purified from either wild-type (WT) or MyD88−/− B6 donors on day 0 following lethal total body irradiation (TBI, 12Gy) delivered in two doses at 3-hour intervals. BALB/c recipients were transplanted with 5×106 TCD-BM cells from WT B6 donors plus 1×106 T cells purified from either WT or MyD88−/− B6 donors on day 0 following 6 Gy TBI. Isolation of T cells and TCD were performed using a Pan T cell Isolation kit II and anti-CD90-MicroBeads, respectively, and the autoMACS Pro system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Mice were housed in sterilized microisolator cages and received autoclaved hyperchlorinated drinking water for the first three weeks after BMT, and filtered water thereafter.
Automacs pro system
Manufactured by Miltenyi Biotec
Sourced in Germany
The AutoMACS Pro system is a fully automated magnetic cell separation instrument designed for the isolation of target cells from heterogeneous cell populations. The system utilizes magnetic beads coated with antibodies that bind to specific cell surface markers, allowing for the isolation and purification of the desired cell type.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Anti cd90 microbeads, by Miltenyi Biotec (1 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Gentlemacs tissue dissociator, by Miltenyi Biotec (1 mentions)
Anti mouse igg1 microbeads, by Miltenyi Biotec (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Facsverse, by BD (1 mentions)
Facslyric, by BD (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase a, by Roche (1 mentions)
Fibrinogen, by Hyphen Biomed (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Cd41 fitc, by BD (1 mentions)
Af555 β tubulin, by Cell Signaling Technology (1 mentions)
Stemspan sfem, by STEMCELL (1 mentions)
Fc block, by BD (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Cd14 pe antibody, by Immunotools (1 mentions)
12 protocols using automacs pro system
1
Allogeneic Hematopoietic Cell Transplantation
2
Immune Cell Profiling of PVRIG Knockout Mice
3
Bovine PBMC Isolation and CD14+ Enrichment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bovine peripheral blood mononuclear cells (PBMCs) were purified from blood samples by density gradient centrifugation using Percoll (GE Healthcare, Little Chalfont, UK). CD14+ cells were freshly isolated from PBMCs using the autoMACS Pro System (Miltenyi Biotec, Bergisch Gladbach, Germany) with anti-bovine CD14 mAb (CAM36A, Washington State University Monoclonal Antibody Center, Pullman, WA, USA), and anti-mouse IgG1 MicroBeads (Miltenyi Biotec) as described previously with some modification (14 (link)). CD14− PBMCs were prepared from negative fractions of CD14+ cell sorting. The purity of each cell fraction was confirmed using FACS Verse (BD Biosciences) or FACS Lyric (BD Biosciences). Highly pure populations (>95%) were used for experiments.
4
Microglia/Macrophage Isolation from Retina
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The retinas were digested with 1.5 mg/mL collagenase A (Roche, Basel, Switzerland) and 0.4 mg/mL DNase I (Sigma-Aldrich) for 30 min at 37 °C in HBSS. Single-cell suspensions were generated by passing the solution through 70 μm filters. Sorting was performed using an autoMACS Pro system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Briefly, to purify microglia/macrophages (MG/MΦ), single-cell suspension was incubated with anti-CD11b-microbeads.
5
Megakaryocyte Spreading Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow cells were lineage (B220−Mac-1−Gr-1−CD16/32−) depleted on an AutoMACS Pro system (Miltenyi) and suspended in StemSpan SFEM (StemCell Technologies) supplemented with 2.6% fetal bovine serum, 1% L-glutamine and stem cell factor (20 ng/mL) then cultured for 2 days at 37°C in 5% CO2. Cells were transferred into fresh medium containing thrombopoietin (50 ng/mL) and cultured for 4 more days. Mature megakaryocytes were enriched on a bovine serum albumin gradient as previously described15 (link) and plated in 12-well μ-Chamber glass slides (ibidi) coated with fibronectin (500 μg/mL; Life Technology), fibrinogen (100 μg/mL; Hyphen BioMed) or collagen (1:20; StemCell Technologies). Cells were then allowed to spread for 3–8 h. Cells were fixed in μ-Chamber slides with 4% formaldehyde/phosphate-buffered saline, permeabilized with 0.1% Triton-X100/phosphate-buffered saline and blocked with FcBlock (1:500; BD Biosciences), then labeled with FITC-CD41 (BD Biosciences) and AF555-β-tubulin (Cell Signaling) antibodies or AF555-Phalloidin (Molecular Probes). All conditions for both controls and knockouts were applied to the same μ-Chamber slide to minimize variation between samples.
6
PBMC Isolation and pDC Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMC were separated from whole blood by Pancoll (PAN-Biotech, Cat# P04-60500) density gradient centrifugation. Sorting was performed using autoMACS Pro System (Miltenyi Biotech) according to manufacturer's instructions. PBMC were stained with CD14-PE antibody (Immunotools, Cat# 21620144, clone 18D11) followed by incubation with anti-PE microbeads (Miltenyi Cat# 130-048-801). The depleted fraction was collected and resuspended in R10 medium – i.e., RPMI 1640 (Sigma-Aldrich) containing 10% heat inactivated FBS (Sigma-Aldrich), 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate, 100 U/ml non-essential amino-acids, 50 μM 2-mercaptoethanol (Invitrogen). 5 × 105 cells/well were seeded in 96-well plates and stimulated with 20 µg/ml cGAMP or 1 µg/ml R848 (Invivogen France). For pDC depletion, PBMCs were stained with PE conjugated anti-BDCA4 (BioLegend Cat# 354504, RRID:AB_11219194) and anti-CD123 (BD Biosciences Cat# 555644, RRID:AB_396001) antibodies, and then sorted using anti-PE microbeads (Miltenyi) as above. Mock-depleted or pDC-depleted PBMCs were stimulated with R848 or ODN M362. Cell depletion was checked by flow cytometry on LSRII cytometer (BD Biosciences, RRID:SCR_002159). Supernatants were collected at 24 h and tested for IFNα by ELISA as described above.
7
Profiling CD8+ Tumor-Infiltrating T Cells
8
Neutrophil Chemotaxis Assay Using Transwell
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils were isolated from bone marrow of hind legs of R223 and Q223 mice. Bones were cleaned and ground in sterile HBSS, then cells were filtered through 100-µm and 40-µm cell filters. Anti-Ly6G magnetically activated cell sorting bead separation was performed according to the manufacturer’s protocol (Miltenyi Biotec) on an AutoMACS Pro system. A total of 4 × 104 neutrophils were immediately added to the top of the transwell filter. A 600-µl volume of HBSS (plus Ca and Mg) containing 10 ng leptin, 50 ng leptin, or 10 ng CXCL1 and CXCL2 (5 ng each; Peprotech) was added to the bottom well. Chemotaxis was allowed to proceed for 1.5 h at 37°C. Filters were then removed from the plates, and excess liquid was removed from the top of the filter with a cotton-tipped applicator. Filters were cut out, fixed with methanol, and then stained with crystal violet. Neutrophils were counted on one field of view with 20× magnification by using an Olympus DP71.
9
Isolation and Stimulation of Murine Monocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For bone marrow extraction, both intact femurs of the animals were rapidly extracted after euthanasia and muscle tissue was removed before further processing. Bone marrow cavity was opened under laminar flow on both sides and the bone was flushed several times with RPMI1640 medium (Thermo Fisher Scientific, Waltham, USA) using a 25G cannula. Cells were passed through a 70μm sieve before flow cytometry and cell isolation.
Monocytes were negatively isolated from bone marrow cells by depletion of non-target cells via labelling with antibodies linked to magnetic beads (Monocyte Isolation Kit (BM)) according to the manufacturer’s instructions using an automatised AutoMACS Pro system (both Miltenyi Biotech, Bergisch Gladbach, Germany). Purity of isolation was checked by flow cytometry and reached >90% CD11b+ Ly6C+ cells.
For in vitro stimulation, cells were resuspended into RPMI1640 supplemented with 10% FBS, placed into a 96-well microplate (50.000 monocytes/well), and stimulated with 100ng/ml ultrapure lipopolysaccharide (LPS, E.coli 0111:B4), 250μg/ml zymosan (Zymosan depleted) (both Invivogen, San Diego, USA) or mock for 24h. TNF-α and IL-6 supernatant levels were quantified by DuoSet ELISA (Bio-Teche, Minneapolis, USA).
Monocytes were negatively isolated from bone marrow cells by depletion of non-target cells via labelling with antibodies linked to magnetic beads (Monocyte Isolation Kit (BM)) according to the manufacturer’s instructions using an automatised AutoMACS Pro system (both Miltenyi Biotech, Bergisch Gladbach, Germany). Purity of isolation was checked by flow cytometry and reached >90% CD11b+ Ly6C+ cells.
For in vitro stimulation, cells were resuspended into RPMI1640 supplemented with 10% FBS, placed into a 96-well microplate (50.000 monocytes/well), and stimulated with 100ng/ml ultrapure lipopolysaccharide (LPS, E.coli 0111:B4), 250μg/ml zymosan (Zymosan depleted) (both Invivogen, San Diego, USA) or mock for 24h. TNF-α and IL-6 supernatant levels were quantified by DuoSet ELISA (Bio-Teche, Minneapolis, USA).
10
Generation of Monocyte-Derived Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monocytes were isolated from peripheral blood mononuclear cells from human volunteer donors by negative magnetic separation using the Monocyte Isolation Kit II (MACS Miltenyi #130-091-153, Miltenyi Biotech, Bergisch Gladbach, Germany) and the AutoMACS pro system (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. In order to generate MDMs, monocytes were resuspended in 100 mL of macrophage media. Cells were cultured in an incubator at 37 °C at 5% CO2 for 6–7 days before using for experiments. All experiments with blood from human volunteer donors were approved by the blood donation service from Boehringer Ingelheim Pharma GmbH and Co. KG following ethical standards and local regulation.
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!