Naïve CD4 and CD8 T cells were isolated from PBMCs using EasySep isolation kits (StemCell Technologies). RNA was extracted using RNeasy Plus micro Kit (QIAGEN) and cDNA generated using VILO mastermix (Thermo Fisher Scientific). Total CD45 (gene name: PTPRC) expression was quantified using a Taqman assay (Thermo Fisher Scientific). CD45 isoforms were determined using optimized RT-PCR primers developed by Fukuhara et al. (2002) (link). In brief, cDNA input was normalized to total CD45 expression for each sample. Normalized input was used in a 30 cycles RT-PCR using the P3 and P7 primers (Fukuhara et al., 2002 (link)). Equal amounts of RT-PCR products were ran on an agarose gel for visualization and quantified by densitometry in ImageJ.
Easysep isolation kits
Manufactured by STEMCELL
Sourced in Canada
EasySep isolation kits are a series of cell separation products designed to isolate various cell types from complex biological samples. The kits utilize magnetic particles and customized protocols to selectively label and isolate the target cell population. The core function of the EasySep kits is to provide a simple, fast, and efficient method for isolating specific cells from a heterogeneous sample.
Automatically generated - may contain errors
Lab products found in correlation
Taqman assay, by Thermo Fisher Scientific (2 mentions)
Vilo master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Abi prism 7900 ht detection system, by Thermo Fisher Scientific (1 mentions)
Complete rpmi 1640, by Lonza (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Foxp3 antibody, by Thermo Fisher Scientific (1 mentions)
Ifn γ antibody, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
α il 4, by Thermo Fisher Scientific (1 mentions)
Human tgf β, by R&D Systems (1 mentions)
Human b cll cell isolation kit, by Miltenyi Biotec (1 mentions)
Ficoll paque gradient, by GE Healthcare (1 mentions)
Anti pe conjugated microbeads, by Miltenyi Biotec (1 mentions)
5 protocols using easysep isolation kits
1
CD45 Isoform Analysis in T Cells
2
Isolation and Gene Expression Analysis of Naïve T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Naïve CD4 and CD8 T cells were isolated fresh PBMCs using EasySep isolation kits (StemCell Technologies). RNA and cDNA was generated as described above. Gene expression was quantified using Taqman assays (Thermo Fisher Scientific) on an ABI Prism 7900HT Detection System (Applied Biosystems), and normalized to RPLP0 as a control gene. Taqman assays used in these studies are listed in Supplementary Table S3 .
3
Adoptive T-cell Transfer in Murine Gammaherpesvirus Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes were isolated from mice at 2 mo post-infection and pooled from multiple mice. Splenocytes were negatively selected for CD4, CD8, or total T cells using EasySep isolation kits (Catalog Nos. 19765, 19853, and 19851; STEMCELL Technologies Inc., Vancouver, BC, Canada). Negative selection was confirmed by flow cytometry analysis to >90% purity. Three million cells in 100 μL were injected into the tail vein of each B6.SJL-PtprcaPepcb/BoyJ mouse. Twenty-four hours after T-cell transfer, the recipient mice were intranasally challenged with 5 × 103 PFU WT MHV-68. Spleens were harvested 14 d post-challenge for the infectious center assay and flow cytometry to confirm the presence of donor T-cells in the recipient mice with anti-CD45.1 and anti-CD45.2.
4
Murine T Cell Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified α-CD3 (145-2C11), purified α-CD28, α-CD25 (3C7), Biotin α-CD25, fluorescein isothiocyanate (FITC)-α-CD4, phycoerythrin (PE)-α-CD25, unconjugated, (allophycocyanin) APC, Alexafluor-647 or PE conjugated α-CD44, APC, Alexafluor 647, PE or PE-Cy7 conjugated IL-17-A, Foxp3 antibodies, IL-6 blocking antibodies, α-IL-4 and IFN-γ antibodies, were all purchased from eBiosciences (San Diego, CA). Mouse CD4 isolation kit II was purchased from Miltenyi Biotec (Auburn, CA). EasySep isolation kits were purchased from Stem Cell technologies (Canada). Human IL-2 purchased from NCI, was a kind gift from Mike Lenardo lab (NIAID, NIH). Recombinant mouse IL-6 was purchased from BioBasic Inc (Amherst, NY), and human TGF-β was purchased from R&D Systems (Minneapolis, MN). Mouse cells were cultured in complete RPMI-1640 (Bio-Whittaker) supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 5 mM Glutamax (Invitrogen) and 50 μM β-mercaptoethanol.
5
Isolation and Purification of Immune Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The peripheral blood mononuclear cells (PBMCs) and BM mononuclear cells were isolated using Ficoll-Paque gradients (GE Healthcare). CD8+ or CD3+ T cells were isolated by negative selection using the Easysep isolation kits (Stem Cell Technologies) with a purity of >90% (online supplemental figure 1a, b ). For effector T cell (CD8+CCR7-) isolation, CD8+ T cells were stained with phycoerythrin (PE)-conjugated anti-CCR7 antibody, followed by anti-PE-conjugated microbeads (Miltenyi) with a purity of >90% (online supplemental figure 1c ). B-CLLs were isolated using the human B-CLL Cell Isolation Kit (Miltenyi) with a purity of >90% (online supplemental figure 1d ).
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!