AIM-V media and CD4+ and CD8+ Dynal beads were purchased from Invitrogen. RPMI-1640 media and X-Vivo 15 media (BioWhittaker™) were from Lonza Biologics, Inc. Pooled Human AB Serum (PHS) was from Valley Biomedical. Ficoll-Paque Premium was from GE Healthcare Sciences. Aldrithiol-2 (AT-2) and reduced L-Glutathione were from Sigma-Aldrich. Good manufacturing practice-certified (GMP-certified) Interleukin-2 (IL-2), GMP-certified Interleukin-4 (IL-4), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), GMP-certified Anti-CD3 antibody, CliniMACS CD4 and CD14 microbeads, CliniMACS LS Tubing sets, CryoMACS DMSO and CliniMACS PBS/EDTA buffer were from Miltenyi Biotec, Inc. Polyinosinic-polycytidylic acid – poly-L-lysine carboxymethylcellulose (Poly-ICLC) was from Oncovir, Inc. Fluorescent antibodies for IL-2, -IFNγ, -TNFα, -MIP-1β, -CD3, -CD4, and -CD8 were purchased from BD biosciences. Antibodies for CD86, -CD14, -CD11c, and -HLA-DR were purchased from Biolegend. Recombinant human IL-2 and IL-7 were from R&D Systems. The following antibodies were manufactured in-house at the AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory: anti-p24CA [94–0001], anti-p17MA [R193244], anti-gp120HIV [P3F5-D5-F8]. Anti-gp41TM(4E10) was obtained from Polymun [AB004].
Clinimacs pbs edta buffer
Manufactured by Miltenyi Biotec
Sourced in Germany
The CliniMACS PBS/EDTA buffer is a sterile, ready-to-use solution designed for cell separation procedures using the CliniMACS system. It is a phosphate-buffered saline solution with EDTA, which helps maintain the integrity and viability of the cells during the separation process.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytopermtm kit, by BD (2 mentions)
Aim 5 media, by Thermo Fisher Scientific (1 mentions)
Mip 1β, by BD (1 mentions)
Anti cd3 antibody, by Miltenyi Biotec (1 mentions)
Cryomacs dmso, by Miltenyi Biotec (1 mentions)
Tnf α, by BD (1 mentions)
Rpmi 1640 media, by Lonza (1 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cryostor cs10, by Merck Group (1 mentions)
Clinimacs plus instrument, by Miltenyi Biotec (1 mentions)
Poch 100i automated hematology analyzer, by Sysmex (1 mentions)
Vi cell xr, by Beckman Coulter (1 mentions)
Fortessa, by BD (1 mentions)
Nucleocounter nc 200, by ChemoMetec (1 mentions)
Macsquant x, by Miltenyi Biotec (1 mentions)
Cd3 fitc, by Miltenyi Biotec (1 mentions)
Cd8 apc vio770, by Miltenyi Biotec (1 mentions)
Cd137 apc, by Miltenyi Biotec (1 mentions)
Cd25 pe vio770, by Miltenyi Biotec (1 mentions)
12 protocols using clinimacs pbs edta buffer
1
Reagents and Antibodies for T-cell Culture
2
Multiparametric Flow Cytometry of Frozen T Cells
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The majority of the extracellular flow cytometry experiments were performed directly on fresh cells, but for six donors, the T cell markers CD69, CD25, CXCR3, CCR6, CD38, HLA-DR, CD127, and PD-1 were analyzed on paired frozen samples. Staining was carried out in 96-well plates with ≤1 × 106 cells/well in 50 μl CliniMACS PBS/EDTA buffer (Miltenyi Biotech, Bergish Gladbach, Germany) supplemented with 0.1% bovine serum albumin. The cells were incubated with mAbs for 30 min at 4°C. Intracellular staining was performed after extracellular staining using the BD Cytofix/Cytoperm™ kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's instructions. 7AAD staining was used to sort live and dead cells if no intracellular staining was performed. The antibodies used in this study are listed in Supplementary Table 1 available online at https://doi.org/10.1155/2017/8010961 . Data was collected using a BD FACSCanto flow cytometer and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Results from subgating were only included if the parent population consisted of ≥80 cells.
3
Isolation and Cryopreservation of CD3+ T Cells
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CD3+ cells were purified from healthy donors’ Leukopaks received from Hemacare (Northridge, CA, USA). Upon reception, the leukapheresis material was analyzed using the Sysmex pocH-100i Automated Hematology Analyzer (Sysmex, Milton Keynes, UK), diluted with CliniMACS PBS/EDTA buffer (Miltenyi, Bisley, UK), supplemented with 0.5% human AB serum (Seralab, Hayward Heath, UK) and centrifuged at 300 × g for 15 min at room temperature to remove platelets. The CD3+ fraction was purified from the plasma on the CliniMACS Plus instrument (Miltenyi) using the CliniMACS CD4 and CD8 reagents (Miltenyi) anti-CD4 and anti-CD8 monoclonal antibodies conjugated to superparamagnetic iron dextran particles. The viability of the final CD3+ product was assessed using the Vi-CELL XR (Beckman Coulter, High Wycombe, UK) and Nucleocounter NC-200 (ChemoMetec, Allerod, Denmark). The number of CD45+ cells, CD3+ cells, CD4+ cells, and CD8+ cells was evaluated before and after the cell separation on the CliniMACS by flow cytometry on the Fortessa (BD Biosciences, San Jose, CA, USA). The acceptance criteria for the CD3+ cell purification were set as follows: (1) viability over 95% and (2) number of CD3+ cells over 95%. Following purification, a working cell bank of CD3+ cells was cryopreserved in CryoStor CS10 (Sigma, Gillingham, UK) until use.
4
Multiparametric Flow Cytometry Analysis
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Data were acquired on a MACSQuant® Analyzer 10 or MACSQuant® X (Miltenyi Biotec) and analyzed with FlowLogic™ V.8 (Inivai Technologies) by either frequency or median fluorescence intensity (MFI). Cells were stained in CliniMACS® PBS/EDTA Buffer (Miltenyi Biotec, catalog no.: 200-070-025) with 0.5% BSA at 4°C for 10 min. Antibodies used for flow cytometry: CD137-APC (clone: REA765, Miltenyi Biotec), CD25-PE-Vio770 (clone: REA570, Miltenyi Biotec), CD69-VioBlue (clone: REA824, Miltenyi Biotec), LNGFR-PE (clone: REA844, Miltenyi Biotec), CD4-VioGreen (clone: REA623), CD8-APC-Vio770 (clone: REA734, Miltenyi Biotec), CD3-FITC (clone: REA613, Miltenyi Biotec), CD45-VioBlue (clone: REA747, Miltenyi Biotec), CD279-PE-Vio770 (clone: REA1165, Miltenyi Biotec), CD366-APC (clone: REA635, Miltenyi Biotec), CD223-FITC (clone: REA351, Miltenyi Biotec). Antibodies were used according to the manufacturer’s protocol.
5
Automated Treg Expansion and Formulation
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A customized process for fully automated expansion ExpAct Treg bead removal and final formulation in 0.9% NaCl/1%HA (Human Albumin 200 g/l, Baxter) infusion solution using the CliniMACS Prodigy® system was developed together with Miltenyi Biotec and successively optimized. Treg culture in the CentriCult unit, bead removal over the column and final formulation were performed with a single CliniMACS Prodigy TS 510 tubing set. The final optimized process includes multiple washing steps to exchange the media to infusion solution, after which the cells pass the primed magnetic column in a two-stage process. The final product is harvested to a sealable cell bag (Cell differentiation bag, Miltenyi Biotec). ExpAct bead removal using the CliniMACS® Plus system was performed following the manufacturer's recommendations to compare the newly developed CliniMACS Prodigy® bead removal process with the standard procedure. Briefly, CliniMACS® Plus bead removal was performed using the CliniMACS® PBS/EDTA buffer (Miltenyi Biotec) supplemented with 0.5%HA (200 g/L, Baxter), the CliniMACS® Tubing Set LS (Miltenyi Biotec) and the software sequence Depletion 2.1.
6
Multiparametric Flow Cytometry Analysis of T-cell Phenotypes
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A list for the antibodies used in this study is shown in Supplementary Table S1 . Extracellular staining was performed in CliniMACS PBS/EDTA buffer (Miltenyi Biotech cat no. 200-070-025) supplemented with 0.1% bovine serum albumin. Extracellular markers were analyzed on viable cells (7-AAD negative). Intracellular staining of α-SMA, IFN-γ, and TNF-α was performed using the BD Cytofix/CytopermTM kit (BD Biosciences, cat no. 554714) according to the manufacturer's instructions. Intracellular staining for FOXP3 was done with FOXP3 staining buffer set (eBioscience, cat no. 0052300) according to the manufacturer's instructions. Phycoerythrin (PE) conjugated CD107a was added in the cell cultures together with PMA/I, Brefeldin A, and GolgiStop on day 5 for 6 h. Boolean gating strategy was used to study the frequency of CD4+ and CD8+ T-cells that were triple positive for TIM-3, PD-1 and CTLA-4 or for TIM-3, PD-1, and LAG-3. A FACSCanto (BD) was used for data acquisition and FlowJo (Tree Star, Ashland, OR, USA) version 10.2 was used for data analysis. Sub-gating for all analyzed samples was done with more than 100 events, and no data points were excluded due to low events.
7
Analysis of Mucosal-Associated Invariant T Cells
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IVB and PB samples were analyzed in pairs, either fresh or thawed after freezing. Frozen cells were thawed in complete medium, washed in PBS and counted, and fresh cells were used directly after isolation. The cells were plated in a 96-well plate, ≤ 1 × 106 cells/well. Staining was carried out in 50 μL of CliniMACS PBS/EDTA buffer (Miltenyi Biotech, Bergish Gladbach, Germany) supplemented with 0.1% bovine serum albumin (FACS-buffer) to which antibodies were added, and incubated for 30 min at 4°C. After washing, the cells were stained with 7AAD, which was used to distinguish live from dead cells. The MR1 tetramers were produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne, and the MR1 tetramer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie. The flow cytometry antibodies used in this study are listed in Supplementary Table S1 . Data were collected using a BD FACSCanto flow cytometer and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Data on conventional T cells were analyzed after excluding CD161+ and Vα7.2+ T cells.
8
Multiparameter Flow Cytometry Analysis
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Extracellular staining was performed in CliniMACS PBS/EDTA buffer (Miltenyi Biotech) supplemented with 0.1% bovine serum albumin. Intracellular staining was performed following extracellular staining using the BD Cytofix/CytopermTM kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s instructions. When only analysing extracellular markers, 7AAD was used to distinguish between live and dead cells. A list of the antibodies used in this study is shown in Supplemental Table 1 . Data was collected using a BD FACSCanto flow cytometer and analysed with FlowJo software (Tree Star, Ashland, OR). Sub-gating was only performed when the parent gate contained more than 80 cells.
9
Isolation and Characterization of Cancer Stem Cells
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Cells were mechanically dissociated to obtain single cell suspensions. After centrifugation cells were resuspended in CliniMACS PBS/EDTA buffer (Miltenyi Biotec) with 0,5 % human serum (PAA). Before staining with fluorochrome conjugated antibodies, Fc receptors were blocked with FcR Blocking Reagent (Miltenyi Biotec). Antibody staining was conducted with CD133/1 and CD133/2 (Miltenyi Biotec, clones AC133 and 293C3) and anti-CD15-antibody (BD, clone MMA) according to the manufacturer’s protocols. Acquisition was performed on a FACS Canto II (BD Biosciences). Dead cells were excluded by 7-AAD (BD Biosciences) staining. Expression of aldehyde dehydrogenase (ALDH) was examined using the ALDEFLUOR kit (STEMCELL Technologies) according to the manufacturer’s protocol.
10
CD4/CD8 Positive Selection from Leukopak
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Using Sepax C-pro, the fresh leukopak was washed and concentrated using CliniMACS PBS/EDTA Buffer (Miltenyi Biotech) supplemented with 0.1% HSA and then incubated with antibodies specific to CD4 and CD8 (Miltenyi Biotec) at room temperature for 30 minutes. Labeled leukopak cells were then transferred on CliniMACS Plus (Miltenyi Biotech) and positive fraction of flow through was collected according to manufacturers' instructions.
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