Viably frozen PBMCs obtained from 8 macaques prior to immunization, post-2nd Ad5hr-recombinant immunization, and 2 wpi and 12 wpi were thawed and stained (1 ul 5-OP-RU tetramer-PE/1 million cells) for 30 minutes at RT, magnetically labeled with anti-PE magnetic beads (Miltenyi Biotec), and positively isolated using a magnetic column. This subset was considered enriched MAIT cells (Fig. 4 ). MR1− cells were labeled with anti-CD3 magnetic beads (Miltenyi Biotec), isolated using a magnetic column and considered CD3+MR1− T cells. MR1−CD3− cells were labeled with anti-CD19 magnetic beads (Miltenyi Biotec), isolated using a magnetic column and considered B cells. To check the purity of the enriched MAIT cells, PBMC obtained prior to immunization from 5 additional macaques were stained with 5-OP-RU tetramer conjugated with PE (NIH Tetramer Core Facility) for 30 minutes; followed by Alexa700 anti-CD3 (SP34-2), BV711 anti-CD4 (L200), BV650 anti-CD8 (RPA-T8), APC-Cy7 anti-CD20 (2H7), BUV395 anti-CD123 (7G3), BV421 anti-CD11b (ICRF44) BV786 anti-CD45 (D058-1283) all from BD Biosciences, FITC anti-CD66abce (TET2) from Miltenyi Biotec, PE-Cy7 anti-NKG2A (Z199) from Beckman Coulter. The cells were acquired on a Symphony (BD Biosciences) and analyzed as described above.
Anti cd19 magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD19 magnetic beads are a lab equipment product designed for the isolation and enrichment of CD19-positive cells. The beads are conjugated with antibodies specific to the CD19 surface marker, allowing for the separation of CD19-expressing cells from a heterogeneous cell population using a magnetic field.
Automatically generated - may contain errors
Lab products found in correlation
Anti human igm f ab 2, by Jackson ImmunoResearch (3 mentions)
Anti human ig a g m f ab 2, by Jackson ImmunoResearch (3 mentions)
Anti cd3 magnetic bead, by Miltenyi Biotec (2 mentions)
Pe cy7 anti nkg2a z199, by Beckman Coulter (1 mentions)
Anti pe magnetic beads, by Miltenyi Biotec (1 mentions)
Q vd oph, by Merck Group (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Accucount 5.27 μm blank particles, by Spherotech (1 mentions)
Anti mouse igg microbeads, by Miltenyi Biotec (1 mentions)
Cd11c magnetic beads, by Miltenyi Biotec (1 mentions)
Phytohemagglutinin (pha), by Thermo Fisher Scientific (1 mentions)
Anti cd8 pe cy7, by BioLegend (1 mentions)
Percp cy5.5 anti cd4, by BioLegend (1 mentions)
Easysep rhesus cd4 t cell isolation kit, by STEMCELL (1 mentions)
Pe cy5 anti cd19 j3 119, by Beckman Coulter (1 mentions)
Lsr 2, by BD (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Anti cd4 magnetic beads, by Miltenyi Biotec (1 mentions)
Automacs sorter, by Miltenyi Biotec (1 mentions)
Cell strainer, by Corning (1 mentions)
17 protocols using anti cd19 magnetic beads
1
Enrichment and Characterization of MAIT Cells
2
Proliferation of Rag1-/- pro-B cells
3
B Cell Activation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated or thawed PBMC were washed with complete medium and counted. For qPCR, B cells were positively selected using anti-CD19 magnetic beads (Miltenyi Biotech). 0.5–1.0 × 106 cells per well were seeded in round-bottom 96-well plates in 200μL 10% FBS/RPMI 1640. Cells were stimulated with anti-human IgM F(ab′2) or anti-human Ig(A + G + M) F(ab′2) (Jackson Immuno-Research Laboratories) for indicated times. After stimulation, cells were collected for flow cytometry or qPCR.
4
Isolation of Purified B Cells and APCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WBCs were harvested as described and were first depleted of T cells by anti-CD3 magnetic beads (Miltenyi Biotec). B cells were then positively selected for using anti-CD19 magnetic beads (Miltenyi Biotec). The unbound cells containing non–B cell APCs were subjected to a second round of CD19 selection to remove the residual B cells. Flow cytometry analysis showed high (>95%) purity of the B cells and the non–B cell APCs (monocytes, pDCs, and DCs).
5
Isolation of Mouse B Cells, DCs, and LMΦ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
B cells were isolated from mouse spleens using anti-CD19 magnetic beads according to the manufacturer’s instructions. In brief, the cells were incubated with anti-CD19 magnetic beads for 15 min on ice and washed with 1 ml of MACS buffer (solution containing phosphate-buffered saline [PBS], pH 7.2, 0.5% bovine serum albumin [BSA], and 2 mM EDTA) (Miltenyi Biotec, Bergisch Gladbach, Germany). The cellular suspensions collected were washed twice in MACS buffer and passed through a magnetic column. Then, CD19+ B cells were isolated by positive selection, washed, resuspended in complete RPMI 1640 medium, and counted before coculture with DCs or lung macrophages (LMφ), as described below.
From the mononuclear cells isolated from the mouse lungs, spleen, and bone marrow, CD11c+ DCs were isolated using CD11c magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD64+ LMφ were isolated using a CD64 primary antibody (clone X54-5/7.1; BioLegend, CA, USA) and anti-mouse IgG microbeads (Miltenyi Biotec), as described for B cell isolation.
From the mononuclear cells isolated from the mouse lungs, spleen, and bone marrow, CD11c+ DCs were isolated using CD11c magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD64+ LMφ were isolated using a CD64 primary antibody (clone X54-5/7.1; BioLegend, CA, USA) and anti-mouse IgG microbeads (Miltenyi Biotec), as described for B cell isolation.
6
SARS-CoV-2 and HCoV-specific B cell responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 2~3x106 PBMCs were enriched for B cells through anti-CD19 magnetic beads (Miltenyi), flow through cells were centrifuged and incubated with 1 μg/mL peptide megapools consisting of SARS-CoV-2 spike, SARS-CoV-2 remainder (SARS-CoV-2 proteome minus spike), HCoVs (HKU1, OC43, NL63, 229E) spike, HCoVs remainder (HCoVs proteome minus spike), and cytomegalovirus (CMV), as well as phytohemagglutinin (500x, Thermo fisher) and DMSO. Cells were cultured in 96 well round-bottom plates with RPMI containing 10% FBS for 24 hours. Cells were stained with Biolegend antibodies FITC anti-CD27 (356404), PE anti-CCR7 (353204), Percp cy5.5 anti-CD4 (357414), PE-cy7 anti-CD8 (344712), APC anti-OX40 (350008), APC-Cy7 anti-CD45RA (304128), AF700 anti-CD3 (300424), BV510 anti-CD19 (302242), BV510 anti-CD14 (367124), BV605 anti-CD69 (310938), BV711 anti-CD137(309832).
7
B Cell Activation and Analysis
8
Isolation and Characterization of Primate Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Viably frozen PBMCs obtained from three macaques prior to immunization, 14 infected macaques 40 weeks post-infection, and nine protected macaques 2 weeks following the last challenge were thawed and used for either negative selection of CD4+ T cells (~ 20–40 million cells) or positive selection of B cells (~ 10–20 million cells). CD4+ T cells were negatively sorted using the EasySep Rhesus CD4+ T cell isolation kit (STEMCELL, Cambridge, MA), stained with anti-CXCR5-PE (Nonhuman Primate Reagent Resource), treated with anti-PE microbeads (Miltenyi Biotech) and positively isolated using a magnetic column. For B cell positive selection, cells were labeled with anti-CD19 magnetic beads (Miltenyi Biotec) and isolated using a magnetic column. To check the purity of the enriched cTFH cells and B cells obtained by this procedure, PBMC obtained from three additional necropsied macaques were stained with Alexa700 anti-CD3 (SP34-2), BV711 anti-CD4 (L200), FITC anti-CD8 (RPA-T8) (BD Bioscience), BV650 anti-CD20 (2H7) (BioLegend), BV605 anti-CD16 (CB16) (eBioscience), and PE-Cy5 anti-CD19 (J3-119) from Beckman Coulter. The cells were acquired on a BD LSRII (BD Biosciences) and analyzed as described above.
9
Adoptive Transfer of Immune Cells in SARS-CoV Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes were isolated from SARS-CoV-infected (9 dpi; sensitized) or naïve BALB/c mice (over 6 months old); the resulting cells then were administered (at 4×107 cells/animal) to recipient naïve SCID mouse i.v. 1 day before challenge. Splenocytes from NK cell-depleted BALB/c mice also were administered to recipient NK cell-depleted SCID mice as described above. CD4+ cells (1×107 cells) were isolated from the spleen of naïve BALB/c mice using anti-CD4 magnetic beads, followed by purification with an AutoMACS sorter (Miltenyi Biotech, Bergisch Gladbach, Germany). Isolated CD4+ cells (>96% purity) were adoptively transplanted into each recipient naïve SCID or nude mouse. In addition, splenocytes of naïve BALB/c mice were divided into the CD19+ cells (B cells) and residual cells (CD19− cells, including T cells) using anti-CD19 magnetic beads (Miltenyi Biotech). The B cells (CD19+cells; 2×107 cells/mouse) and/or the residual cells (CD19− cells; 2×107 cells/mouse) then were adoptively transplanted into the recipient naïve SCID mice.
10
Isolation and Enrichment of PBMC and Synovial Tissue Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMC) were prepared from citrated blood samples using Ficoll® Paque Plus density centrifugation following manufacturer's instructions (GE Healthcare). Synovial tissue was dissected followed by digestion for 2 h at 37°C in 1 mg ml−1 Collagenase 1 (Sigma-Aldrich). Debris was removed and cells isolated by sequentially passing through 100, 70 and 40 μm cell strainers (Corning). PBMC and synovial tissue were enriched for B-cells using either anti-CD19 magnetic beads or anti-CD20 (Miltenyi Biotech) as outlined in Supplementary Table 5 .
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!