Hi Geraldine, 

Lois Salamonson in Australia may be able to answer this question.

Hope you're well and look forward to perhaps seeing you in Switzerland next July at the time of ESHRE.  If a free space was available, I could arrange a good public educational event, especially in Geneva, since so many good experts will be there.  Any suggestions?

Suggestions for the location: not really.

There is a Congress center.

Dear Gealdine

Which protocols have been used so far?

Dear Gealdine

sample was treated with 1× BD Pharm Lyse buffer (BD Pharmingen, San Jose, CA, USA) for 15 min at room temperature to remove RBCs and washed twice in phosphate-buffered saline (PBS).

Dilute with cold buffer(PBS or HBSS) with a ratio of one to three

Use a ratio of one to one or one to two cold ficole

Around 410g centrifuges at a temperature of 22 ° C.

Blood in the tube is very important. Should be done slowly. It is better to slightly tilt tube.

Please note the attached protocol (2).

[The yield of mononuclear cells from blood depends on many factors

including the percentage of contaminating granulocytes and platelets and efficiency of erythrocyte removal (Thong et al., 1983). However, the most critical step is the under layer of Ficoll-Hypaque. A distinct and clear fraction between the Ficoll-Hypaque and leukocyte/RBC PBS mixture must be seen prior to centrifugation. If not, clear separation of the leukocyte layer will not occur and the mononuclear cell population may be lost. For maximum yield and purity after centrifugation,

it is also essential to remove all the material at the Ficoll-Hypaque interface and to ensure that no Ficoll-Hypaque solution or supernatant is removed with the sample. Including parts of the Ficoll-Hypaque layer will increase the granulocyte contamination; including supernatant will increase the platelet contamination.

Erythrocytes can aggregate and trap lymphocytes in the clumps. These clumps will

sediment into the pellet and reduce the yield of lymphocytes. Diluting the blood with

PBS before Ficoll-Hypaque centrifugationwill reduce the clumping, as well as get rid of platelet contamination. In addition, the temperature of the centrifuge can affect the yield of lymphocytes. At low temperatures, lymphocyte yields are reduced because a longer centrifugation time is required. At high temperatures,

lymphocyte viability is decreased and erythrocyte aggregation is increased. During

the gradient centrifugation, the temperature should be maintained at 18◦ to 20◦C.

Occasionally, it is necessary to isolate mononuclear cells from clotted blood. This

has been successfully accomplished by using streptokinase to dissolve the blood clot. Lymphocytes isolated form clotted blood function in some respects similarly to those isolated from heparinized blood, although the lymphocyte yield is only 60% of the yield from heparinized blood (Niku et al., 1987).]

Best

saeed

Many thanks Saeed.

Hi Geraldine

Hypotonic lysis is a gentler process than acid lysis to remove RBCs and still preserve the remaining blood elements as best as possible.  I have not worked with menstrual blood before, but I presume hypotonic lysis would work just as well as it does for peripheral blood, umbilical cord blood, etc.

Bicarbonate-buffered, ammonium chloride RBC lysis solution: 0.15 M NH4Cl, 0.01 M NaHCO3, 1.0 mM EDTA. 

Use fresh buffer at room temperature.  You need to use a high v/v of buffer/blood to achieve thorough lysis. Incubate for 15 mins at room temperature, then wash the cells twice in an isotonic buffer (e.g. PBS).

Hope this helps.  Regards, Rosemary 

Dear Geraldine Canny , 

Though i haven't worked with menstrual blood sample, but i have successfully removed RBC from Blood, spleen , Liver  and Bone marrow of Mice sample. I have used ACK lysis buffer for many times and now, i'm using ' Lympholyte ' to separate RBC and WBC , & it's working fine. If you need detailed protocol , just knock me for once.