Blood Cells Isolation/Activation and RNA Purification Procedures
Cells were isolated either from whole blood treated with CPDA-1 anticoagulant or from platelet-depleted buffy coats, both obtained from normal healthy donors tested negative for HIV-I, HIV-II, hepatitis B and syphilis, the ratio male\ female being approximately 2:1.
Erythrocytes were removed by the treatment of whole blood or buffy coats with Dextran T500 (Pharmacia, Biotech) (1), plasma being used as a source of platelets in the former case. White blood cells were concentrated by the centrifugation, and mononuclear cells (MN) (B- and T-lymphocytes and monocytes) and polymorphonuclear cells (PMN) (mostly neutrophils) were separated in a preformed Percoll (Pharmacia) gradient (2).
Platelets were isolated as described (3, 4).
The purity of MN, PMN and platelets was more than 98% for each cell population, as it was evaluated by staining fixed smears of freshly isolated cells with hematoxylin.
Resting CD14+ (monocytes), CD4+ (T helper \ inducer cells), CD8+ (T supressor \ cytotoxic cells) and CD19+ (B lymphocytes) were positively selected from MN cell suspensions by immunomagnetic separation using Dynabeads M-450 according to Dynal recommendations ( 5). The purity of CD4+, CD8+ and CD19+ was not less than 95% for each cell population, as it was evaluated by immunostaining fixed smears of positively isolated target cells, detached from Dynabeads using the corresponding DETACHaBEAD systems ( 5). The immunostaining was performed by a routine procedure using primary antibodies to CD4, CD8 and CD19 ( Becton & Dickinson), Vectastatin ABC Kit ( Vector Lab.) and DAB ( Sigma) as a substrate according to the standard Vectastatin ABC Kit protocol.
Activation of MN cells, B- and T-lymphocytes was performed as recommended (6, 7 and 8) with some modifications. Isolated cells were suspended in RPMI 1640 containing 10% fetal calf serum ( Gibco), 100 U\ml penicillin, 100 mg\ml streptomycin, 2 mM glutamine, vitamins, non-essential aminoacids and sodium pyruvate ( High Clone) , the cell density was adjusted up to 1´ 10 6 cell \ ml, and the cultures were incubated at 37oC in a humidified atmosphere of 5% CO2 in air.
Activation of lymphocytes was estimated on the basis of morphological criteria ( blast morphology, mitoses) and expression of the two activation markers, CD25 ( IL-2 receptor) and CD71 ( transferrin receptor), established immunocytochemically as pointed above.
In the case of MN cells the culture medium was supplemented with 2 ml\ml PWM (pokeweed mitogen, Gibco) and 5 mg\ml Con A (concanavalin A, ICN ), and the cultures were incubated for 3 days. The activated cells were collected, washed and used for isolation of total RNA. The immunocytochemical analysis showed that the preparations of activated MN cells contained 51±12% CD4+, 42±14% CD8+, 6±4% CD19+ and 60±17% CD25+.
Activated CD4+ cells ( CD4+ blasts) were isolated in a discontinuous Percoll gradient after CD8+, CD19+ and partially CD14+ - depleted MN cells had been incubated with 5 mg\ml Con A for 3-4 days. The preparations of activated CD4+ contained 90±4% CD4+, 2±1% CD8+, 94±3% CD25+ . There were no observed CD19+ cells.
Activated CD8+ cells were positively selected by immunomagnetic separation from CD14+ and CD4+ - depleted MN cells incubated with 5 mg\ml PHA (phytohemagglutinin, Pan Eco, Russia ) for 3 days. The preparations of CD8+ activated cells contained 95±3% CD8+, 1±1% CD4+, 2±1% CD19+ and 88±1% CD25+.
Activated CD19+ cells were positevely selected by immunomagnetic separation from CD14+ and CD8+ - depleted MN cells incubated with 2ml\ml PWM for 4 days. The preparations of activated CD19+ cells contained 92±2% CD19+, 4±2% CD4+ and 87±3% CD71+. There were no observed CD8+ cells.
References
1. Scandinavian Journal of Immunology, 1976, Supplement n 5 : Lymphocytes. Isolation, fractionation and characterization. Edited by J.B.Natvig, P.Perlmann & H. Wigzell
2. Percoll. Methodology and applications. Pharmacia Fine Chemicals
3. A.V.Mazurov et al. Thrombos. Haemostas. 1991, 66, 494-499
4. A.I.Kuznetsov et al. Eur.J. Haematol. 1992, 49, 113-118
5. Cell separation and protein purification. Technical Handbook, Second Edition. Dynal
6. Lymphocytes. A practical approach, 1987. Edited by G.G.B.Klaus. IRL Press
7. Fundamental Immunology, 1984. Editor W.E.Paul. Raven Press
8. G.Sieber et al. Blut, 1980, 41, 81-92
Total RNA was isolated using Qiagen RNeasy Midi Kit followed by DNAase treatment ( RNAase-free DNAase, Epicentre ) with subsequent cleaning up RNA preparations using repeated Qiagen RNeasy Midi Kit procedure. All preparations had the value of 28S/18S ribosomal RNAs ratio no less than 1.6 . QC was made using Agilent BioAnalyzer. Typical QC results are shown here.
PolyA+RNA was isolated from Total RNA using two repeated steps of oligo (dT) chromatography, and was considered to be passed QC if no genomic DNA was detected after 40 cycles of Real-Time PCR using MHC Class I primers.
Figure Typical results on glyoxal electrophoresis of isolated RNA preparations.
L1. RNA Ladder
1. PolyA+ RNA, A549
2. PolyA+ RNA, HeLa
3. PolyA+ RNA, G361
4. Total RNA, A549
5. Total RNA, HeLa
6. Total RNA, G361
L2. 1 kb RNA Ladder
