Separation of mouse spleen cells by passage through columns of sephadex G-10

Ly, Iris A.; Mishell, Robert I.

doi:10.1016/0022-1759(74)90108-2

Cited by 592 publications

(179 citation statements)

References 9 publications

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“…However, in addition to memory B cells, nylon wool also specifically retains AFC's and macrophages (Handwerger and Schwartz, 1974). These latter two cell populations can be depleted selectively from cell suspensions by Sephadex G-10 fractionation (Ly and Mishell, 1974).…”

Section: Discussionmentioning

confidence: 99%

“…As the AFC has been implicated in the adoptive transfer of immunity to Plasmodium berghei infections in mice (Gravely and Kreier, 1976), its possible role in the protection against B. microti infections observed after the transfer of immune spleen cells was investigated. Separation of spleen cells on Sephadex G-10 columns has been shown to specifically remove AFC's together with macrophages (Ly and Mishell, 1974). Therefore, Sephadex G-10 adherent and non-adherent immune spleen cells were tested for their protective effect after transfer.…”

Section: Introductionmentioning

confidence: 99%

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BABESIA MICROTI IN MICE. SUBPOPULATIONS OF CELLS INVOLVED IN THE ADOPTIVE TRANSFER OF IMMUNITY WITH IMMUNE SPLEEN CELLS

Meeusen

Lloyd

Soulsby

1984

Aust J Exp Biol Med

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Summary Protection against a primary Babesia microti infection in mice, induced by the adoptive transfer of immune spleen cells, was abolished when the immune spleen cells were treated with mitomycin C prior to transfer. Since mitomycin C treatment prevents the replication of lymphocytes without affecting other cell functions, these results would suggest that the transferred cells required proliferation in the recipient mice before they could exert their protective effect, and this excludes the already differentiated antibody‐forming cells (AFC's), macrophages and sensitised helper T cells. This was partly supported by the finding that Sephadex G‐10 non‐adherent immune cells, depleted of macrophages and AFC's, still conferred a strong protection after transfer. However, the Sephadex G‐10 adherent cells, on a cell to cell basis, initially conferred a better protection against B. microti than did the non‐adherent cells or unfractionated immune spleen cells. The possibility of the retention of an intermediate B memory cell type on the Sephadex G‐10 columns and the suppression of antibody production are discussed in view of these results.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

BABESIA MICROTI IN MICE. SUBPOPULATIONS OF CELLS INVOLVED IN THE ADOPTIVE TRANSFER OF IMMUNITY WITH IMMUNE SPLEEN CELLS

Meeusen

Lloyd

Soulsby

1984

Aust J Exp Biol Med

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“…Spleen cells from in vivo primed mice were depleted of B cells and adherent cells (APC) by a double pas sage through Sephadex G-10 columns (Pharmacia Fine Chemicals, Uppsala, Sweden) (Ly et al, 1974) followed by a nylonwool passage (Scrubbed Nylon Fiber, Ferwall Laboratories, Deerfield, IL, USA) as described by Kast et al, (1988). FACS analysis showed that the responder cells con tained > 70% T cells (data not shown).…”

Section: Apc Activity O F the Ce Fractionsmentioning

confidence: 99%

A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation

Ossevoort

Toes

Bruijn

et al. 1992

Journal of Immunological Methods

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The standard isolation procedure for antigen presenting dendritic cells (DC) takes 2 days and includes selective adherence to tissue culture plates which may lead to the activation of these cells. This report describes the isolation of DC by centrifugal elutriation (CE). Murine spleen cells were separated on the basis of size and density into 7 CE fractions. This method took 90 min. Ceils from each CE fraction were characterized by fluorescence activated cell sorter (FACS) analysis and their antigen presenting cell (APC) activity was determined by a secondary Sendai virus specific T cell proliferation assay. CE fraction 5 contained most of the DC with a concentration of 6-10% , representing an approximately 15-fold enrichment compared to unseparated spleen cells ( < 1 % DC). This CE fraction also exhibited the highest APC activity, which was almost completely abolished after depletion of DC by treatment with monoclonal antibody 33D1 (DC-marker) and complement. Further enrichment of CE fraction 5 by discontinuous density gradient centrifugation resulted in a cell population containing 35-55% 33Dl-positive cells with similar characteristics as DC isolated by the standard procedure, such as the capacity to induce a primary viral peptide specific CTL response. Two-color FACS analysis showed an increase in MHC expression on 33Dl-positive cells of CE fraction 5 after 18 h culture involving cell adhesion to a similar level as the M HC expression on DC isolated by the standard procedure. During this same period their morphology changed from a round to a dendritic appearance. In conclusion, our results indicate that CE is well suited for isolating DC more rapidly and without activation of these cells by adherence, a process which readily occurs in the standard isolation procedure.

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“…Mononuclear cells were separated from fresh heparinized venous blood and fresh heparinized bone marrow aspirates by FicollPaque (Pharmacial Fine Chemicals Inc., Uppsala, Sweden) density gradient centrifugation (Boyum 1968). Monocytes were removed by passing the mononuclear cells through a Sephadex G-10 column (Pharmacia Fine Chemicals Inc., Piscataway, N.J., USA) according to the method of Ly and Mishell (1974). Separation of monocyte-depleted mononuclear cells into E-rosette forming cells and non-E-rosette forming cells was carried out by rosetting with neuraminidase-treated sheep erythrocytes (SRBC) (Weiner et al 1973).…”

Section: Methodsmentioning

confidence: 99%

Ecto-5'-nucleotidase activities in lymphocytes and B lymphoblastoid cell lines from patients with congenital agammaglobulinemia.

Nakae

Tsuchiya

Okumura

et al. 1983

Tohoku J. Exp. Med.

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Separation of mouse spleen cells by passage through columns of sephadex G-10

Cited by 592 publications

References 9 publications

BABESIA MICROTI IN MICE. SUBPOPULATIONS OF CELLS INVOLVED IN THE ADOPTIVE TRANSFER OF IMMUNITY WITH IMMUNE SPLEEN CELLS

BABESIA MICROTI IN MICE. SUBPOPULATIONS OF CELLS INVOLVED IN THE ADOPTIVE TRANSFER OF IMMUNITY WITH IMMUNE SPLEEN CELLS

A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation

Ecto-5'-nucleotidase activities in lymphocytes and B lymphoblastoid cell lines from patients with congenital agammaglobulinemia.

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