Histone peptides H2B (10PKKGSKKAVTKAQKKDGKKRKRSR33), H3 (91QSSAVMALQEASEAY105), and H4 (71TYTEHAKRKTVTAMDVVYALKRQ93), Spliceosomal peptide (SmB 136GPSQQVMTPQGRGTVAAA153), and U1 small nuclear ribonucleoprotein of 70kDa (U1 70K 131RIHMVYSKRSGKPRGYAFIEY151) were used in the non-phosphorylated form (GL Biochem, Shanghai, China). Peptides were at least 90 % pure as determined by reverse phase HPLC and Maldi-T of mass spectroscopy and dissolved in 5 % DMSO/endotoxin-free Hank’s balanced saline solution with phenol red (HBSS). Each peptide was each added to cell cultures at 5, 10, and 20 μg/ml, unless stated otherwise. Additionally, cells were stimulated with anti-CD3 mAb (OKT-3, 2 μg/ml), tuberculin purified protein derivative (PPD; Statens Serum Institut, Copenhagen, Denmark; 5 μg/ml), and Staphylococcal enterotoxin B (SEB; Sigma-Aldrich, Poole, Dorset, UK; 0.5 μg/ml).
Tuberculin purified protein derivative
Manufactured by Statens Serum Institut
Sourced in Denmark
Tuberculin purified protein derivative (PPD) is a diagnostic tool used to detect exposure to Mycobacterium tuberculosis, the bacteria that causes tuberculosis. It is a sterile, purified protein extract derived from the culture filtrate of M. tuberculosis. When administered through an intradermal injection, PPD elicits a delayed-type hypersensitivity reaction in individuals who have been previously exposed to the tuberculosis bacteria, indicating the presence of cell-mediated immunity.
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5 protocols using tuberculin purified protein derivative
1
Histone and Spliceosomal Peptide Stimulation
2
Purification of Cut4 and CFP21 Mycobacterial Proteins
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Cut4 (Rv3452) and CFP21 (Rv1984c) were produced as N-terminal His-tag fusion proteins as previously described [6 (link), 31 (link)]. Briefly, genes were amplified from cosmids MTCY13E12 and MTCY39, respectively (Pasteur Institute, Paris, France), and cloned into pDEST™14 expression vector allowing the expression of N-terminal His-tag fusion proteins. All proteins were produced in E. coli strains, refolded from inclusion bodies and purified using a Ni2+-NTA column and gel filtration [31 (link)].
For CFP21, the His-tag was removed by enzymatic digestion with TEV and proteins without His-tag were purified by exclusion from a Ni2+-NTA column and conserved in Tris 10 mM, NaCl 150 mM, pH 8. Due to its peculiar pH instability and NaCl sensitivity, the Cut4 His-tag was not removed and protein was conserved into 50 mM CHES buffer, pH 9 [31 (link)]. Tuberculin Purified Protein Derivative (PPD) was purchased from Statens Serum Institut (Denmark).
For CFP21, the His-tag was removed by enzymatic digestion with TEV and proteins without His-tag were purified by exclusion from a Ni2+-NTA column and conserved in Tris 10 mM, NaCl 150 mM, pH 8. Due to its peculiar pH instability and NaCl sensitivity, the Cut4 His-tag was not removed and protein was conserved into 50 mM CHES buffer, pH 9 [31 (link)]. Tuberculin Purified Protein Derivative (PPD) was purchased from Statens Serum Institut (Denmark).
3
Onchocerca volvulus and Brugia malayi Antigens
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A soluble extract of Onchocerca volvulus worm antigen (OV) was prepared as previously described [20] (link) and extracts of Brugia malayi female worms were prepared from infected jirds that were or were not treated with tetracycline (BmFE and BmFEtet, [38] (link)). Recombinant peptidoglycan-associated lipoprotein (wPAL) and Wolbachia surface protein (WSP) were prepared as previously described [38] (link), [39] (link). LPS (Serratia marescens) was obtained from Sigma-Aldrich (Taufkirchen, Germany) and tuberculin purified protein derivative (PPD) from Statens Serum Institute (Copenhagen, Denmark). Anti-CD3 and anti-CD28 antibodies were purchased from eBiosciences (Frankfurt, Germany). Polymyxin B sulphate salt (P4932) and secondary antibodies for IgG1-4 were obtained from Sigma-Aldrich, whereas that for IgE was purchased from Southern Biotech (Birmingham, USA).
4
PBMC Isolation and Stimulation Protocol
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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors (NHS National Blood Service, Bristol, UK) or from patients with congenital HA, with a current negative FVIII inhibitor status, at the Haemophilia Treatment Center at Radboud University Medical Center (Nijmegen, The Netherlands), as previously described.60 (link) All studies were performed with the approval of the local ethics committees and after informed consent was obtained from the patients and healthy volunteers. PBMCs were stimulated with 5 to 10 µg/mL rhFVIII, 20 to 40 µg/mL peptides, or 20 µg/mL tuberculin purified protein derivative (PPD) (Statens Serum Institute, Copenhagen, Denmark) as a positive control, and proliferation was measured as previously described.35 (link)
5
Profiling HPV16-Specific T-Cell Responses
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Eight peptide pools of HPV16 peptides were used to determine the proliferative capacity of HPV16-specific T-cells, and the cytokine polarization and frequency of HPV16-specific Foxp3+ regulatory T-cells, as described previously [15 (link), 37 (link), 39 (link)]. 30 (or 35) amino-acid-long peptides with 14- (or 15-)mer overlap covering the HPV16 E2, E6, and E7 protein sequences were synthesized by the solid-phase peptide synthesis (SPPS) method with >95% purity (ChinaPeptides Co., Shanghai, China). Two pools of E2 peptides (E2.1 and E2.2) consisted of 12 or 11 (30-mer) peptides, respectively. Four pools of E6 (E6.1–E6.4) and two pools of E7 peptides (E7.1 and E7.2) consisted of two 32-mer or 35-mer peptides, respectively [15 (link)]. Memory response mix (MRM) stock solution (50×), consisting of tetanus toxoid, 0.75 fL/mL (Statens Serum Institut, Copenhagen, Denmark), tuberculin purified protein derivative (PPD), 5 μg/mL (Statens Serum Institut), and Candida albicans, 0.015% (Greer Laboratories, Lenoir, NC, USA) was used as a positive control for the proliferation assays and cytokine production capacity of the PBMCs [15 (link), 39 (link)].
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