Following 30 minute incubation with primary antibody and centrifugation at 700g spin for 10 minutes, the nuclei + 1°Ab complex in the pellet was resuspended in 80μl of MACS Buffer composed of 1X PBS (Tissue Culture grade; Ca2+, Mg2+ free), 0.5% Nuclease free Bovine Serum Albumin (BSA), and 2mM EDTA. Next, 20 μl of Anti-Rabbit IgG Microbeads (Miltenyi) was added with fluorophore-conjugated 2°Ab (dilution 1:400), mixed, and incubated for 20 minutes in the refrigerator in the dark. This was followed by a wash with 1ml of MACS buffer at 300g for 10 minutes at 4°C. Subsequently, the recommended protocol for Anti-Rabbit IgG Microbeads-mediated magnetic separation/enrichment of immunolabeled nuclei was performed with MACS MS columns (Miltenyi). Both the flow through (FT) and eluate fractions were collected and mounted with Vectashield + DAPI (Vector Labs) on glass slides to visualize with a confocal microscope for evaluating sensitivity, specificity, and fold enrichment of the MAN-IP assay.
Anti rabbit igg microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-Rabbit IgG Microbeads are paramagnetic beads conjugated with antibodies specific to rabbit immunoglobulin G (IgG). They are designed for the magnetic separation and enrichment of cells, proteins, or other biological targets bound to rabbit IgG.
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Lab products found in correlation
Large cell column, by Miltenyi Biotec (2 mentions)
Lympholyte mammal, by Cedarlane (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Macs ms column, by Miltenyi Biotec (1 mentions)
Vectashield dapi, by Vector Laboratories (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Osteoblast mineralization medium, by PromoCell (1 mentions)
Dispase 2, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerol phosphate, by Merck Group (1 mentions)
Osteoblast growth medium, by PromoCell (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix 2 no ung, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Running buffer, by Miltenyi Biotec (1 mentions)
Ab13840, by Abcam (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
11 protocols using anti rabbit igg microbeads
1
MAN-IP Nuclei Enrichment Workflow
2
Isolation and Sorting of IgG-Expressing B-Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from the ethylenediaminetetraacetic acid (EDTA) containing peripheral blood by density-gradient centrifugation with Lympholyte-Mammal (Cedarlane, Burlington, NC, USA), as described in the manual. The isolated PBMCs (107 cells) were next washed with RPMI (Life Technologies, Carlsbad, CA, USA) containing DNase I (Roche, Basel, Switzerland) and re-suspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Then, the IgG expressing B-cells were isolated using Anti-Rabbit IgG Microbeads (Miltenyi Biotech, Auburn, CA, USA).
The isolated B-cells were stained for viability, incubated with a cocktail of anti-rabbit IgG and fluorochrome-conjugated specific peptide in the dark for 30 min at 4 °C, and washed with ice-cold PBS. Finally, cells were re-suspended in PBS and subjected to Fluorescence Activated Cell Sorting (FACS) analyses. Sorting was carried out using BD Influx Cell Sorter and BD FACS DIVA software (UC Davis Medical Center, Sacramento, CA, USA). Single B-cells expressing IgG were sorted into a 96-well plate (omitting row H).
The isolated B-cells were stained for viability, incubated with a cocktail of anti-rabbit IgG and fluorochrome-conjugated specific peptide in the dark for 30 min at 4 °C, and washed with ice-cold PBS. Finally, cells were re-suspended in PBS and subjected to Fluorescence Activated Cell Sorting (FACS) analyses. Sorting was carried out using BD Influx Cell Sorter and BD FACS DIVA software (UC Davis Medical Center, Sacramento, CA, USA). Single B-cells expressing IgG were sorted into a 96-well plate (omitting row H).
3
Isolation and Culture of Rat Alveolar Epithelial Cells
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The animal study protocols were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (approval number for production: SCXK (PLA) 20120011; approval number for application: SCXK (PLA) 20120031). AECs were obtained through the detailed procedure reported in our previous study.12 (link) Sprague‒Dawley rats weighing 120 – 150 g and aged 3 – 4 weeks were obtained from the animal center of Daping Hospital, Army Medical University. After euthanasia, pharyngeal skin and tissues were dissected to perform tracheal intubation. The lung samples were obtained en bloc from the rats, and were washed twice with saline before being completely digested, cut into pieces, and filtered. The resulting samples were then centrifuged to obtain the cell precipitate, which was dispersed and blended with anti-rat T1α (Sigma, #1995, St. Louis, USA). The cells were subsequently incubated with anti-rabbit IgG microbeads (Miltenyi Biotec, #130-047-102, Bergisch Gladbach, Germany), and then resuspended and cultured in 8-well plates.
4
Isolation and Expansion of Primary Osteoblasts
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Primary osteoblast precursor cells were isolated from the calvarial bone of newborn C57BL/6 mice (1- to 2-day-old) through enzymatic digestion with α-MEM containing 0.1% collagenase (Life technologies) and 0.2% dispase II (Life Technologies). The isolated osteoblast precursor cells were promoted with osteogenic α-MEM medium with 10% FBS, 1% penicillin–streptomycin, 5 mM β-glycerol phosphate (Sigma), 0.1 mg ml−1 ascorbic acid (Sigma) and 10 nM dexamethasone (Sigma) for 9 days and culture medium was replaced every 2–3 days36 (link). The human primary osteoblasts (human OBs) were purchased from PromoCell. The human OB cells were cultured and promoted with osteoblast growth medium and osteoblast mineralization medium (PromoCell), respectively. The purified osteoblasts (ALP+ cells) were isolated by MACS using anti-ALP antibody (Abcam, Rabbit IgG, Ab108337) in combination with anti-Rabbit IgG MicroBeads (Miltenyi Biotec).
5
Isolation and Analysis of Testicular E-cadherin Cells
6
Purification and Characterization of Ddx4+ Cells
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OC pieces were digested by 1 mg/mL collagenase and 1 μg/mL DNAse I (Sigma-Aldrich) for 120 min at 37 °C. Cell pellets were then suspended in running buffer (Miltenyi Biotec) and incubated with 10 µL of rabbit anti-human Ddx4 antibody for 30 min at 4 °C. Thus, the samples were treated with anti-rabbit IgG microbeads (Miltenyi Biotec), and Ddx4+cells were isolated by an automated magnetic-activated cell sorting system (autoMACS Pro, Miltenyi Biotec,) as previously described [11 (link)]. The purity of cell population was assessed by the flow cytometry detection of membrane Ddx4. Briefly, an aliquot of isolated cells was incubated with rabbit anti-human Ddx4 antibody (Abcam ab13840) and then processed with an FITC-conjugated anti-rabbit antibody (Sigma-Aldrich). In addition, cytoplasmic expression of Ddx4 was also evaluated by flow cytometry, after cell fixation and permeabilization as described for A2780 cells. In each case, an isotype control was used as negative parameter.
7
Optimizing Bead-Based Cardiac Nuclei Isolation
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COS7 (Catalog #ATCC CRL-1651, African green monkey kidney fibroblast-like cell line) was used for performing experiments to validate and use the optimally performing beads. Protein G Dynabeads (ThermoFisher Scientific, #10003D) did not perform with high specificity and sensitivity as previously reported17 when used with tagged cardiac nuclei. Therefore, Anti-Rabbit IgG Microbeads (Miltenyi) were used for all experiments due to its superior performance.
8
Isolation of Schwann Cells from Mouse Adipose
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From N=5 BTBR ob/ob and N=5 BTBR wild type mice, bilateral inguinal subcutaneous white adipose depots were pooled for each animal. Tissues were placed in an enzyme digest containing 2mg/mL collagenase A (Sigma-Aldrich, Cat#10103586001), 2mg/mL dispase II (Sigmam Cat#D4693) , and 0.05% Trypsin (Gibco, Cat#25200056). Tissues were dissociated for 30 minutes at 37°C using a gentleMACS Octo Dissociator with Heaters (Miltenyi, Cat#130096427). Cells were passed through a 100μm filter and spun down to pellet stromal vascular fraction (SVF). Cells were resuspended in buffer containing anti-CD45 microbeads (Miltenyi, Cat#130052301) and incubated for 15 minutes at 4°C. Cells were washed and incubated for 10 minutes at room temperature with anti-Pref1 antibody (Cell Signaling, Cat#2069) followed by incubation with anti-Rabbit IgG microbeads (Miltenyi, Cat#130048602) for 15 minutes at 4°C. Cells were passed through Large cell columns (Miltenyi, Cat# 130042202) in a magnetic field and unlabeled flow-through (Cd45-, Pref-1-) was collected. Collected cells were incubated with anti-O4 microbeads (Miltenyi, Cat#130096670). Cells were passed through a Large cell column (Miltenyi, Cat# 130042202) in a magnetic field and O4+ Schwann cells were collected from column in 300 μl of Trizol.
9
Magnetic Sorting of Myocyte Nuclei
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Magnetic-assisted cell sorting (MACS) of myocyte nuclei was performed after isolation and staining of nuclei with a PCM1 antibody (1:1,000, HPA023370, Sigma). Nuclei were centrifuged and resuspended in 80 μl MACS buffer. Anti-rabbit IgG MicroBeads (20 μl; Miltenyi, Bergisch Gladbach, Germany) were added and incubated for 15 min at 4 °C. MACS buffer (900 μl) was added and the nuclei suspension was applied to an M column (Miltenyi), washed with 3 ml MACS buffer and eluted with 1 ml MACS buffer after removal of the magnet. The eluate was applied to another M column, washed again and eluted in 1 ml elution buffer (1 mM EDTA in PBS). Sorting efficiency was determined by staining aliquots before and after cell separation with an Alexa488-labelled secondary anti-rabbit antibody (1:1,000, Invitrogen) and by flow cytometric analysis (CyFlow Space, Partec).
10
Yeast Display Library Induction and Sorting
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To induce the SNU475 yeast display library, the cell population grown in SD-CAA medium was moved to SG-CAA medium (SD-CAA with 2% galactose instead of dextrose) and incubated at 20°C for 40 h. In 100 µl of MACS buffer (PBSM; 0.8% NaCl, 0.02% KCl, 0.144% Na 2 HPO 4 , 0.024% KH 2 PO 4 , 0.5% BSA, and 0.0744% EDTA-2Na), 3 × 10 7 induced cells were added and labeled using 5 µg of polyclonal rabbit anti-actin (Invitrogen) at RT for 30 min. After washing with PBSM, 20 µl of anti-rabbit IgG Microbeads (MiltenyiBiotec GmbH, Gladbach, Germany) and 80 µl of resuspended cells were incubated at 4°C for 30 min. After washing again, 3 × 10 7 cells in 500 µl of PBSM were loaded onto the LS column (MiltenyiBiotec GmbH, Gladbach, Germany) surrounded by magnet and washed with 10 ml of PBSM. MAC-sorted cells were incubated in 4 ml of SD-CAA medium with 100 µl of YPD at 30°C for 2 days [4] .
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