Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of male and female blood donors (Transfusion Medicine, University Medicine Greifswald, Germany) using standard Ficoll gradient centrifugation. CD4+ cells and CD8+ cells were positively selected using Dynal CD4 Positive Isolation Kit and Dynal CD8 Positive Isolation Kit (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer’s instructions. All cells were freshly isolated directly before their use. No cryopreserved cells were used in any experiment.
Dynal cd8 positive isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom
The Dynal CD8 Positive Isolation Kit is a tool used to isolate and enrich CD8+ T cells from biological samples. It utilizes magnetic beads coated with antibodies that specifically bind to CD8 surface markers on cells, allowing for the separation and collection of the CD8+ cell population.
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4 protocols using dynal cd8 positive isolation kit
1
PBMC Isolation and CD4/CD8 Cell Separation
2
Isolation of CD8+ T Cells from PBMCs
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Peripheral blood mononuclear cells (PBMCs) were purified from the venous heparinized blood samples of healthy controls and SSc patients by centrifugation on Ficoll-Hypaque gradient (Biochrom AG, Berlin, Germany) for 30 min at 1,800 rpm. CD8+ T lymphocytes were isolated by sequential cycles of cell sorting on magnetic beads using microbeads conjugated with a mAb specific for the CD8 antigen (Dynal CD8 positive isolation kit, Invitrogen by Life Technologies Ltd., Paisley, UK) following the manufacturer’s instructions.
3
PBMC Stimulation and Cytokine Quantification
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Cryopreserved PBMC were thawed in complete media. A portion of the PBMC was depleted of CD8+T cells (CD8depl PBMC) using the Dynal CD8 Positive Isolation Kit (Invitrogen, Carlsbad CA). Total PBMC or CD8depl PBMC were plated in the presence of soluble Leishmania antigens from L. major parasites (SLA, 2.5 μg/mL, generous gift of Dr. Frank Neva) for 6 days at 37°C, 5% CO2. Pokeweed mitogen (PWM, 5 μg/mL, Sigma) was used as a positive control. Cell-free supernatant was collected from each well, triplicate subjects pooled, and used to quantify cytokines using the Q-Plex Human Cytokine–IR Array (Quansys Biosciences, Logan, UT) according to manufacturer’s protocol [23 (link)]. For LPA, cells were pulsed as previously reported [24 (link)].
4
Isolation and Expansion of Antigen-Specific CTLs
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PBMC from buffy coats of three healthy middle-aged HLA-A2.1 donors were isolated, cryopreserved, and stored as previously described (28 (link)). CTLs were established in triplicate for each peptide according to described protocol (28 (link)) and cultured for 2 weeks. CD8 T cells were isolated using Dynal® CD8 Positive Isolation Kit (Invitrogen, Carlsbad, CA). RNA isolation was performed using Dynabeads® mRNA DIRECT™ Kit (Invitrogen). cDNA was synthesized immediately after RNA isolation using oligo(dT) as primer and M-MLV (Invitrogen) as reverse transcriptase. The BV19 gene was amplified from cDNA using BV19-specific primer and fluorochrome-labeled TCR CB primer as described (37 ). For Donor C, CTLs for peptides substituted at position 63 were cultured for 3 weeks without following CD8 isolation. For comparison, M158-66 and negative control cultures (no peptide) were performed in the same manner. As described previously (28 (link)) only PCR products with evidenced of focused CDR3 lengths were further examined.
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