Erythrocyte removal kit
During isolation of cells like mononuclear cells from different sources, many times some of erythrocytes remain in the pellet as unwanted effect. These small number of erythrocytes are problem as there is hardly any method to remove them quickly from bone marrow, tissue as well as pathological lesion. Even to isolate the erythrocytes from the solutions containing circulating tumour cell populations from blood, bone marrow as well as lymphnode biopsies. There are groups working on CAR T-cell therapies, removal of all erythrocytes should be needed to have pure population of mononuclear cells. Similarly during stem cells isolation from umbilical cord blood, erythrocytes may be needed to be removed. There are many more examples e.g. from isolation of particular cell population from umbilical cord wall, the erythrocytes needed to be removed. These are examples, where these erythrocytes are needed to be removed. There are also many applications, where user needs intact erythrocytes to conduct the microscopic analysis as in case of erythrocyte invading pathogens like plasmodium, anaplasma, babesia etc. Genekam has developed the method to isolate these intact erythrocytes with magnetic field, hence one can get erythrocyte free pellet to do further analysis. There will be hardly any lysis of erythrocytes because they will be removed as intact cell population. This kit is very simple to be used (just add Genekam solution, keep for a few minutes and remove the erythrocytes with magnetic field).
This kit is called Genekam erythrocytes removal kit. This kit may be also called erythrocyte separation kit. The user applies magnet to remove the treated erythrocytes. This kit comes as 50X solution, which will be added in PBS to make the needed final concentration. This is a sterile product!
It is 50X solution, hence this kit may be divided into small portions to provide to other users in institute.
The properties and applications of this kit along with the procedure (manual) is as follows:
Storage at 4° C
Expiry time: 2 years
During isolation of cells e.g. white blood cells populations like mononuclear cells from blood, spleen, umbilical blood cord, bone marrow etc, there are often a problem that erythrocytes are mixing in the population. Genekam has developed a kit to remove the erythrocytes with magnetic rack. This kit has big applications during the development of followings:
- immunotherapies like CAR T-cells or dendritic cells
- regenerative therapies e.g. isolation of stem cells from umbilical cord blood
- regenerative therapies e.g. isolation of CD34 positive cells
- nucleic acid isolation e.g. to make pellet erhtrocytes free to isolate the RNA or DNA (where erythrocytes cause inhibition or interference) for PCR, flow cytometry, co focal microscopy as well as next generation sequencing
- to isolate the erythrocytes for detecting the erythrocytes infecting pathogens e.g. Plasmodium, Anaplasma, Babesia etc.
- to get the pure cell population from organs like spleen
Genekam Eryhtrocyte removal solution (50X)
- Magnetic rack
- PBS optional
1. Prepare the ready to use solution in washing PBS without any serum or other additive. ( 2ml of Genekam Erythrocyte removal in 98 ml PBS to get ready to use solution because it is 50X): Now make the pellet of isolated cells containing erythrocytes and suspend this pellet in 4-6 ml Genekam erythrocyte removal solution. Keep it at 4° C for 20 to 30 minutes. Now put the tube in magnetic rack and keep it for 3 minutes. (one will observe that erythrocytes are attractive to the wall through magnetic field). Remove the supernatant from the tube containing the cells free from erythrocytes with pipettor carefully. The pellet or spot containing erythrocytes can be seen as a spot on the wall of the tube because the magnet has attracted the treated erythrocytes. There should be no lysis of erythrocytes!
Hint: If user wants to conduct microscope analysis of erythrocytes containing pathogens. The erythrocytes can be collected as separate population. The erythrocytes can be placed on the microscopic slide for further analysis.
2. Now centrifuge the pellet 2000 rpm for 10 minutes to get the pellet containing cells without erythrocytes.
3. Wash the pellet with PBS and centrifuge it as step 2. This pellet can be used for different applications.
Genekam MNC isolator can be used with above said method to isolate mononuclear cells in large quantity directly from blood. These MNC population can be used for to develop CAR T-cell therapies, other theapies as well as cell culturing.
* Research use only *