scholarly journals A simple procedure for the separation of viable blood cells, suitable for long-term in vitro experiments

1974 ◽  
Vol 10 (2) ◽  
pp. 153-166 ◽  
Author(s):  
Fedor Medzihradsky ◽  
Michael J. Marks ◽  
Joan I. Metcalfe
Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.


1992 ◽  
Vol 11 (3) ◽  
pp. 223-228 ◽  
Author(s):  
Cynthia J. Langlois ◽  
Edward J. Calabrese

Simultaneous exposure to chemicals which can oxidize the haemoglobin of the red blood cell to methaemoglobin is common. Although the effects of some of these agents have been documented individually, little research considers the interactive effects. In-vitro experiments on the treated blood of female Dorset sheep assessed the interactive capacity of chlorite, copper and nitrite to affect methaemoglobin formation. All combinations of doses which produced 2.5, 5, 10% methaemoglobin were tested in all possible combinations (a total of 80), as were the controls. This included data on each chemical alone, each two-way combination and the three-way combination. The response is largely additive (the sum of the individual effects) except for one of the two-way interactions, chlorite/nitrite (P < . 01), which showed antagonism. Chlorite may oxidize nitrite which could explain the less-than-additive response. Overall, the result of combining these agents on methaemoglobin was additive.


Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 999-1006
Author(s):  
CE Eastment ◽  
FW Ruscetti

In long-term hamster bone marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months without need for hydrocortisone or adherent stromal elements, which are requirements for bone marrow growth in all other species studied. Only the most primitive erythroid progenitors (BFU-E) are produced in the cultures. Following treatment of the cells with erythropoietin, these progenitor cells undergo differentiation into mature hemoglobinized red blood cells. Concomitant addition of erythropoietin (Epo) and prostaglandin-E1 (PGE1) results in the production of large numbers of maturing red blood cells. In cultures stimulated with Epo and PGE1, as many as 70% of the cells are benzidine-positive, while Epo alone stimulated as many as 45% of the cells to become erythroid. Epo and PGE1 do not have any apparent deleterious effect on the continuous hemopoiesis occurring in these cultures. Under identical conditions, syngeneic adherent cell cultures do not produce any erythroid elements. The development of mature red blood cells from primitive erythroid precursors occurs in the presence of Epo alone and without any apparent need for adherent stromal elements. These cultures provide a useful in vitro model for dissecting the positive and negative signals that regulate erythropoiesis.


2000 ◽  
Vol 28 (12) ◽  
pp. 1470-1480 ◽  
Author(s):  
Ladan Kobari ◽  
Françoise Pflumio ◽  
Marie-Catherine Giarratana ◽  
Xiaxin Li ◽  
Monique Titeux ◽  
...  

1994 ◽  
Vol 13 (4) ◽  
pp. 247-255 ◽  
Author(s):  
T.H.J. Naber ◽  
F. Heymer ◽  
C.J.A. Van Den Hamer ◽  
W.J.M. Van Den Broek ◽  
J.B.M.J. Jansen

2011 ◽  
Vol 57 (2) ◽  
pp. 174-179 ◽  
Author(s):  
M.G. Zykova ◽  
O.M. Ipatova ◽  
V.N. Prozorovskiy ◽  
N.V. Medvedeva ◽  
A.A. Voskresenskaya ◽  
...  

The drug composition based on the plant phospholipids and the antitumor drug doxorubicin (particle size <30 nm) was obtained using original technology elaborated in the Institute of Biomedical Chemistry (Russian Academy of Medical Sciences). In in vitro experiments demonstrated decreased drug association with blood cells for this nanophospholipid form as compared with free doxorubicin. This was accompanied by a with corresponding increase in its plasma level ans also by drug redistribution from plasma protein fraction to high density lipoproteins. Significance of these changes for doxorubicin biodistributon and antitumor activity is discussed.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Nurul Akmal Che Lah ◽  
Robert Gray ◽  
Sonia Trigueros

AbstractWith the long-term goal of developing an ultra-sensitive microcantilever-based biosensor for versatile biomarker detection, new controlled bioreceptor-analytes systems are being explored to overcome the disadvantages of conventional ones. Gold (Au) microwires have been used as a probe to overcome the tolerance problem that occurs in response to changes in environmental conditions. However, the cytotoxicity of Au microwires is still unclear. Here, we examined the cytotoxicity of Au microwires systems using both commercial and as-synthesised Au microwires. In vitro experiments show that commercial Au microwires with an average quoted length of 5.6 µm are highly toxic against Gram-negative Escherichia coli (E. coli) at 50 µg/mL. However, this toxicity is due to the presence of CTAB surfactant not by the microwires. Conversely, the as-synthesised Au microwires show non-cytotoxicity even at the maximum viable concentration (330 µg/mL). These findings may lead to the development of potentially life-saving cytotoxicity-free biosensors for an early diagnostic of potential diseases.


Author(s):  
М.Г. Голубева

Введение. В патогенезе многих заболеваний важную роль играют изменения функции гемостаза и нарушение реологических свойств крови. Поиск новых лекарственных препаратов, влияющих на взаимодействие форменных элементов крови в процессе тромбообразования, является одной из актуальных задач современной гематологии. Цель исследования: сравнение влияния малых регуляторных пептидов, являющихся фрагментами нейрогормонов, на взаимодействие эритроцитов с тромбоцитами при их агрегации под действием адреналина в экспериментах in vitro. Материалы и методы. Исследовали пептиды, представляющие собой С-концевые фрагменты нейрогормонов: Pro-Arg-Gly-NH2 — фрагмент вазопрессина, Pro-Leu-Gly-NH2 — фрагмент окситоцина. В опытах in vitro пептиды в концентрации 10–4–10–6 М добавляли к пулу богатой тромбоцитами плазмы (БТП) крыс или к смеси БТП с эритроцитарной массой, разведенной 1:1000 физиологическим раствором, или к отмытым эритроцитам и тромбоцитам и их смеси, и определяли изменение агрегации под действием адреналина в конечной концентрации 0,02 ммоль/л. Агрегацию эритроцитов и тромбоцитов регистрировали на агрегометре. Результаты. Установлено, что С-концевой фрагмент вазопрессина обладает более ярко выраженным антиагрегантным действием, чем фрагмент окситоцина, причем это было установлено как в БТП, так и при использовании отмытых клеток крови. Ингибирующее действие фрагмента Pro-Arg-Gly-NH2 сохранялось и на фоне предварительного усиления агрегации тромбоцитов эритроцитами. С-концевой фрагмент окситоцина практически не влиял на агрегацию тромбоцитов. Заключение. Использование малых регуляторных пептидов позволяет снижать агрегационный эффект эритроцитов, улучшая тем самым терапию при тромботических осложнениях. Introduction. Hemostasis changes and disturbances of blood rheological properties play the important role in pathogenesis of many diseases. One of the actual problems of modern hematology is new drugs investigation for aff ecting on blood cells interaction during thrombus formation. Aim: to compare the eff ect of small regulatory peptides (fragments of neurohormones) on the interaction of erythrocytes with platelets under their aggregation by adrenaline in in vitro experiments. Materials and methods. We studied 2 peptides (C-terminal fragments of neurohormones): Pro-Arg-Gly-NH2 — fragment of vasopressin, Pro-Leu-Gly-NH2 — fragment of oxytocin. In in vitro experiments we added peptides (in concentration of 10–4–10–6 М) to a pool of platelet-rich rat plasma (PRP) or to a mixture of PRP with erythrocyte mass diluted 1:1000 by saline solution, or to washed erythrocytes and platelets and their mixture, and determined the aggregation changes under adrenalin in a final concentration of 0.02 mmol/L. Erythrocytes and platelets aggregation was recorded by aggregometer. Results. We found that C-terminal fragment of vasopressin has a more pronounced antiplatelet eff ect than the oxytocin fragment, and it was found both in PRP and with the use of washed blood cells. The inhibitory eff ect of Pro-Arg-Gly-NH2 fragment also remained under preliminary increasing of platelet aggregation by erythrocytes. C-terminal fragment of oxytocin practically had no effect on platelet aggregation. Conclusion. Use of small regulatory peptides helps to reduce the aggregation eff ect of erythrocytes, thereby improving therapy of thrombotic complications.


2000 ◽  
Vol 28 (7) ◽  
pp. 47-48
Author(s):  
L. Douay ◽  
L. Kobari ◽  
F. Pflumio ◽  
M.C. Giarratana ◽  
X. Li ◽  
...  

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2273-2273
Author(s):  
Nathalie W.D. Jansen ◽  
Goris Roosendaal ◽  
Marion Wenting ◽  
Herman A.W. Hazewinkel ◽  
Johannes W.J. Bijlsma ◽  
...  

Abstract Purpose Joint bleeds lead to joint destruction. In vitro exposure of human and canine cartilage to blood results in long lasting severe adverse changes in cartilage. An in vivo joint haemorrhage in the canine knee joint demonstrates similar adverse effects although less outspoken and long-lasting. We investigated the clearance rate of blood from canine knee joints as a possible explanation for this discrepancy. Methods Blood was injected into the knee joint of Beagle dogs, either 48h, 24h or 15m before termination. The amount of red and white blood cells present in the joint cavity was determined. Chondrocyte activity and cartilage matrix integrity as well as cartilage destructive activity of synovial tissue were determined biochemically. Additionally, synovial tissue was analyzed by use of histochemistry. Results Fifteen minutes after the injection of autologous blood, the red blood cell count was 5,7*1012/L, comparable to the amount present in whole blood, and gradually decreased (1,6*1012/L at 24 hours) to 0,2*1012/L within 48 hours (less than 5%). The amount of white blood cells increased in the first 24 hours, and was still increased after 48 hours, although less than after 24 hours. The proteoglycan synthesis rate and -release were adversely affected already within 24 hours (−22% and +24% respectively), and these effects were more severe 48 hours post-injection (−34% and +53% resp.). Synovial tissue culture supernatants demonstrate cartilage destructive properties as expressed by an increased release, a decreased synthesis rate, and decreased content of cartilage proteoglycans; increasing with time after the experimental haemorrhage (+207%/+247%; −58%/−62%; −8%/−28% respectively, for 24/48 hours). Evaluation of the synovial tissue revealed at 15 minutes post-injection countless numbers of intact RBC that were almost completely disappared after 48 hours, withonly limited recruitment of macrophages and iron deposition. Conclusions Blood is cleared very rapidly from the canine knee joint, but in that short time span already has adverse effects on both cartilage and synovial tissue. This rapid clearance can play a role in the discrepancy between long-term in vitro and in vivo effects of blood-induced joint damage since more than 10% v/v blood for 48 hours is needed induced to long-term adverse effects in vitro. Irrespectively, blood has devastating effects on articular cartilage very rapidly, and in this respect it is important to prevent (traumatic) joint haemorrhages and if they occur, to treat them properly.


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