Peripheral blood mononuclear cells (PBMC) were extracted using Ficoll-Paque (GE Healthcare) gradient centrifugation. CD8+ T-cells and CD4+ T-cells were extracted from PBMCs by consecutive positive separation using microbeads (CD4+ #130-045-101; CD8+ #130-045-201) and AutoMACS technology (Miltenyi Biotec) according to the manufacturer’s protocol. The purity of extracted CD4+ T-cells was 91–95% and for CD8+ T-cells 88–91%. All cell populations were collected and stored as cell pellets in a −80 °C freezer.
Automacs technology
Manufactured by Miltenyi Biotec
Sourced in Germany
The AutoMACS technology is a fully automated magnetic cell separation system developed by Miltenyi Biotec. It provides high-purity cell isolation by leveraging magnetic bead-based cell labeling and separation. The core function of the AutoMACS system is to automate the process of magnetic cell separation, enabling efficient and reproducible isolation of target cell populations from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Bme rlt plus buffer, by Qiagen (2 mentions)
Cd4 microbeads, by Miltenyi Biotec (2 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Aria 2 cell sorter, by BD (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Anti cd45 percp cy5, by BD (1 mentions)
Rneasy plus mini kit protocol, by Qiagen (1 mentions)
Cd19 microbeads, by Miltenyi Biotec (1 mentions)
Aria 2 sorter, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
Anti ly6g, by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by BD (1 mentions)
Anti human cd14 microbeads, by Miltenyi Biotec (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Win 55 212 2, by Merck Group (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Mirnaeasy kit, by Qiagen (1 mentions)
9 protocols using automacs technology
1
Isolation and Purification of CD4+ and CD8+ T-Cells
2
Retroviral Transduction and CD2 Marker Selection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bicistronic retroviruses coding for CD2 marker alone (pMIC), CD2 followed by full-length human Sp1 (Sp1) or CD2 followed by mutated Sp1 Zn2,3 (mutations of the second and third Zn Fingers) (Zn). CD2 marker allow transduced cells identification as previously described [9 (link), 14 (link)]. Retroviral particles were generated in 293T HEK packaging cells by transfection with Sp1, Sp1 Zn2,3 (Zn), or empty vector (pMIC) plasmid and concentrated with sucrose gradient separation procedure. Retroviral transduction was performed in 6-well plates, and selection of CD2 transduced cells was released at indicated time by magnetic selection using autoMACS technology (Miltenyi Biotec), according to the manufacturer’s protocol. Briefly, transduced cells were incubated 30 min with 4μg/ml PE-conjugated monoclonal Ab against murine CD2 (BD Biosciences) in Phosphate-buffered saline (PBS) containing 0,5% Bovine Serum Albumin (BSA) and 2mM EDTA. Cells were labelled with anti-PE microbeads (Miltenyi Biotec) and CD2 positive cells were magnetically selected by passing through an autoMACS (Miltenyi Biotec). The purity of positive fractions was evaluated by flow cytometry and was routinely above 95%.
3
Isolation and Purification of HA-Specific CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens and MLN were mashed through 70 µm cell strainers and washed with erythrocyte lysis buffer or PBS containing 2% FCS and 2 mM EDTA, respectively. Lymphocytes from the lamina propria of the colon were isolated as described previously [17 (link)]. In brief, colons were rinsed extensively with ice cold PBS and cut into small pieces followed by washing steps in PBS/2 mM EDTA and cell culture media under constant stirring at 37 °C. Afterwards, colons underwent digestion with collagenase IV (Sigma, Bonn, Germany) for 90 min at 37 °C. Afterwards, suspensions were passed through 70 µm cell strainers to obtain single cell suspensions.
CD4+ T cells were enriched from spleens of TCR-HA/Foxp3-GFP mice using autoMACS technology according to the manufacturer’s recommendation (Miltenyi Biotec, Bergisch-Gladbach, Germany). To obtain a pure population of HA-specific CD4+Foxp3- T cells, cells were stained with α-CD4 and an antibody against the HA-specific TCR (6.5) and sorted for CD4+ 6.5+ (HA-specific TCR) Foxp3– (GFP–) T cells on an ARIA II cell sorter (BD Bioscience, Heidelberg, Germany). Purity of sorted cells was ≥95%.
CD4+ T cells were enriched from spleens of TCR-HA/Foxp3-GFP mice using autoMACS technology according to the manufacturer’s recommendation (Miltenyi Biotec, Bergisch-Gladbach, Germany). To obtain a pure population of HA-specific CD4+Foxp3- T cells, cells were stained with α-CD4 and an antibody against the HA-specific TCR (6.5) and sorted for CD4+ 6.5+ (HA-specific TCR) Foxp3– (GFP–) T cells on an ARIA II cell sorter (BD Bioscience, Heidelberg, Germany). Purity of sorted cells was ≥95%.
4
Isolation and Purification of CD4+ Immune Cell Subsets
5
Isolation of Organ-Specific T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions from the lung and liver were obtained by digestion in media containing 5% FCS, and 100 U/mL collagenase IV of clostridium histolyticum (Sigma-Aldrich, Bonn, Germany) or 200 μg/ml collagenase D (Roche, Mannheim, Germany) respectively, at 37 °C for 45 minutes. Samples were pipetted up and down every 5–10 minutes. Afterwards, the remaining tissue was squashed through 70 μm cell strainers and washed with PBS containing 2% FCS and 2 mM EDTA or erythrocyte lysis buffer, respectively. Spleens and lymph nodes were squashed through 70 μm cell strainers, washed with erythrocyte lysis buffer or PBS containing 2% FCS and 2 mM EDTA, respectively. CD4+ T cells were enriched from spleens of TCR-HA transgenic mice using autoMACS technology (Miltenyi Biotech, Bergisch-Gladbach, Germany) according to manufacturer's instructions. HA-specific CD4+ T cells were obtained by staining cells with α-CD4 and an antibody against the HA-specific TCR11 (link) and sorting them by flow cytometry using an ARIA II Sorter (BD Bioscience, Heidelberg, Germany).
6
Neutrophil Chemotaxis Assays: In vitro and In vivo
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For in vitro chemotaxis assays, neutrophils were purified from BALB/c mice bone-marrow, using PE-conjugated anti-Ly6G Ab (BD Biosciences) and autoMACS technology (Miltenyi Biotec), according to manufacturer’s protocol. 5x105 purified neutrophils were incubated for 3 h in transwell chambers with 5μm polycarbonate filters (Costar) in the presence of BaF3-Sp1 inducible clone previously treated (Ctrl) or not (Sp1) with dox for 24 h. Transmigrated neutrophils Ly6G+ were counted by flow cytometry. For in vivo chemotaxis assay, BaF3-Sp1 inducible cells were cultured 18 h in absence of dox and injected in the peritoneal cavity of BALB/c mice. PBS injected mice were used as controls. Six hours after injection, peritoneal washes were performed, cells were harvested and stained with anti-Ly6G (eBioscience) and anti-CD11b (BD Biosciences) Abs. All surface stainings were performed at 4°C in the dark in PBS supplemented with 1% FCS and 0,09% NaN3 (Sigma-Aldrich). Cellular recruitment was evaluated by flow cytometry.
7
Isolation and Differentiation of Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy anonymous donors (Transfusion Centre of Madrid, under the approved code “PODIS09”) by Ficoll Paque density-gradient. Then, monocytes were purified by positive magnetic isolation using anti-human CD14 microbeads and AutoMACS technology (Miltenyi Biotec). To generate hmoDCs, monocytes were seeded at 1x106 cells/mL concentration and differentiated in the presence of 100 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 100 ng/mL IL-4 (Preprotech) for 6 days. To generate WIN-hmoDCs, WIN55,212-2 (Sigma-Aldrich) at a final concentration of 50 nM was added at days 0 and 4 of the hmoDCs differentiation.
8
Isolation of CD56+ CD3- cells from PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In 26 of the subjects evaluated by flow cytometry (14 MBL and 12 CLL), fresh peripheral blood was also subjected to Ficoll density gradient centrifugation. Purified CD56 + CD3 -cells were isolated from PBMCs based on positive selection methods employing immunomagnetic beads (CD3 and CD56 MicroBeads) and autoMACS technology (Miltenyi Biotec, Bergisch Gladbach, Germany) and were stored at -80ºC in 1% BME RLT-plus buffer (QIAGEN). RNA was finally extracted following the RNeasy Plus Mini Kit protocol (QIAGEN).
9
CD4+ Cell Isolation from Peripheral Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood was subjected to Ficoll centrifugation. Purified CD4+ cells were obtained from mononuclear cells employing positive selection immunomagnetic methods (CD4 MicroBeads) and AutoMACS technology (Miltenyi Biotec, Bergisch Gladbach, Germany). RNA was extracted using the miRNAeasy kit (QIAGEN, Venlo, The Netherlands).
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!