An improved method for separation of neutrophils from human blood using methylcellulose

Zhou, Dongying; Stewart, Gwendolyn J.; Kowalska, MA; Niewiarowski, Stefan

doi:10.1016/0049-3848(95)00176-r

Cited by 1 publication

(1 citation statement)

References 5 publications

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“…Human neutrophils were isolated from citrated blood by layering on polymorphonuclear cells (PMN) separation medium (Robbins Scientific Corp., Sunnyvale, Calif.) followed by a 30 min centrifugation at 450 g at room temperature (Zhou et al , 1995). The interface PMN were harvested and diluted with half‐strength PBS/MC (phosphate‐buffered saline supplemented with 0.2% methylcellulose).…”

Section: Methodsmentioning

confidence: 99%

Megakaryocyte precursors, megakaryocytes and platelets express the HIV co‐receptor CXCR4 on their surface: determination of response to stromal‐derived factor‐1 by megakaryocytes and platelets

Kowalska

Ratajczak²,

Hoxie³

et al. 1999

Br J Haematol

133

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Summary. Thrombocytopenia is a late complication of human immunodeficiency virus (HIV) infection. The chemokine receptor CXCR4 has been shown to be a co-receptor for lymphocyte-tropic HIV-1 strains. CXCR4 is also a natural receptor for the chemokine SDF-1. We have previously shown that CXCR1 and CXCR2 are present on megakaryocytes and platelets. Although interleukin-8 (IL-8) and other chemokines that bind to these two receptors do not activate platelets, they are able to inhibit megakaryocytopoiesis, presumably through these receptors. We therefore examined whether CXCR4 is present on developing and mature megakaryocytes and on platelets. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of CXCR4 message. Immature and mature aIIbb3 þ megakaryocytes, and platelets were also positive for CXCR4 by flow cytometric studies using a CXCR4-specific antibody. We then tested whether SDF-1 can affect the biology of these cells. CD34 þ cells and immature aIIbb3 þ cells responded to SDF-1 as indicated by Ca 2þ mobilization and chemotaxis. However, mature megakaryocytes failed to demonstrate either of these responses, in spite of their continued ability to bind 125 I-SDF-1. Further, SDF-1 failed to inhibit megakaryocyte colony growth. Platelets bound 125 I-SDF-1 with a K D similar to the affinity seen for CXCR4 on other cells, yet SDF-1 did not aggregate washed platelets nor augment aggregation by low-dose ADP or thrombin. SDF-1 also failed to stimulate Ca 2þ mobilization, granular release or expression of P-selectin in platelets. Accordingly, although our studies demonstrate that CD34 þ precursors, megakaryocytes and platelets all express CXCR4 and bind SDF-1, biological effects were only demonstrable of SDF-1 on CD34 þ precursors. The potential biological implications of CXCR4 expression on maturing megakaryocytes and platelets in normal individuals and following HIV infection are discussed.

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Section: Methodsmentioning

confidence: 99%