1. Isolation of CLL Cells from Patient Blood Samples
<ol>
	<li>Peripheral blood samples from previously consented CLL patients are received from the clinic in EDTA blood collection tubes, accompanied by the white cell count (WCC). Purify the peripheral blood CLL samples according to the WCC. For WCC &#60; 40 x 106&#160;cells/mL, proceed to step 1.1.1; for WCC &#8805; 40 x 106&#160;cells/mL, proceed to step 1.1.2.
	<ol>
		<li>Pour the contents of all the EDTA blood tubes into a 50 mL conical centrifuge tube and add 50 &#181;L of Human B Cell Enrichment Cocktail per 1 mL of blood. Incubate at room temperature (RT) for 20 min. Proceed to step 1.1.2.</li>
		<li>Dilute the sample at a ratio of 1:1 with RT CLL wash buffer (phosphate-buffered saline (PBS), 0.5% Fetal Bovine Serum (FBS) and 2 mM EDTA).</li>
	</ol>
	</li>
	<li>Aliquot RT density gradient media into an appropriately sized conical centrifuge tube for the sample (10 mL into a 50 mL tube for 30 mL of sample or 4 mL into a 15 mL tube for 10 mL of sample).</li>
	<li>Carefully layer the sample on top of the density gradient media and centrifuge at 400 x&#160;g&#160;for 30 min at RT. 
	NOTE:&#160;Ensure that the centrifuge is at RT before the samples are placed in the centrifuge as a change in temperature will result in poor enrichment of mononuclear cells, and switch off the brake on the centrifuge, as sudden braking can disrupt the liquid interface.</li>
	<li>Gently harvest the white layer of mononuclear cells that collect at the interface of the density gradient media and CLL wash buffer, into a fresh 50 mL conical centrifuge tube using a plastic Pasteur pipette.</li>
	<li>Add 40 mL of CLL wash buffer to the isolated monolayer to wash the cells and centrifuge at 300 x&#160;g&#160;for 10 min at RT.</li>
	<li>Discard the supernatant, resuspend the pellet by flicking the bottom of the tube, then repeat the wash step described in step 1.5.</li>
	<li>Discard the supernatant, resuspend the pellet as described in step 1.6, then resuspend the pellet in a set volume of CLL wash buffer (up to 40 mL, depending on the size of the cell pellet).</li>
	<li>Count the cells using trypan blue and a haemocytometer. Then proceed to flow cytometry to check the purity of CLL cells. 
	NOTE:&#160;At this stage, the CLL cells can be cultured at a concentration of 10 x 106&#160;cells/mL in media to be used in experiments, and/or cryopreserved in 10% dimethyl sulfoxide (DMSO)/FBS for future work at concentrations of up to 100 x 106&#160;cells/vial.</li>
</ol>

<table><tbody><tr><td>15 mL Tube&amp;nbsp;</td><td>Griener Bio one</td><td>188271</td><td></td></tr><tr><td>1.5 mL microcentrifuge Tubes&amp;nbsp;&amp;nbsp;</td><td>Griener Bio one</td><td>616201</td><td></td></tr><tr><td>DMSO&amp;nbsp;</td><td>Sigma</td><td>&amp;nbsp;D2650</td><td></td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>EDS</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher</td><td>10500064</td><td></td></tr><tr><td>Histopaque1077 density gradient media&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>H8889</td><td></td></tr><tr><td>Trypan Blue Solution&amp;nbsp;</td><td>Thermofisher</td><td>15250061</td><td></td></tr><tr><td>PBS Tablets&amp;nbsp;</td><td>Fisher Scientific</td><td>BR0014G</td><td></td></tr><tr><td>RosetteSep Human B Cell Enrichment Cocktail&amp;nbsp;</td><td>Stem Cell Technologies</td><td>15064</td><td></td></tr><tr><td>Sigma 3-16P&amp;nbsp;&amp;nbsp;</td><td>SciQuip</td><td></td><td>Centrifuge</td></tr></tbody></table>

density gradient centrifugation: a method to isolate cll cells from peripheral blood

Source: Hay, J. et al. <a href="https://www.jove.com/t/60426">Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking.</a> J. Vis. Exp. (2019).
This video describes the technique of isolating chronic lymphocytic leukemia (CLL) cells from peripheral blood. With this protocol, we will isolate CLL cells from human patient blood sample for subsequent efficient generation of nuclear and cytoplasmic fractions.

Density Gradient Centrifugation: A Method to Isolate CLL Cells from Peripheral Blood

Source: Hay, J. et al. Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking. J. Vis. ...

- In chronic lymphocytic leukemia, or CLL patients, B cell lymphocytes significantly increase in the bone marrow and blood. To isolate these CLL cells from a blood sample, first, treat the sample with a human B cell enrichment cocktail that separates B cells using negative selection. The antibodies in the cocktail specifically recognize and cross-link the antigens expressed by non B cells and erythrocytes, forming dense complexes.
Next, layer the sample over a density gradient medium having a density optimal for lymphocyte isolation. With subsequent centrifugation, the different blood components separate based on variations in their densities relative to the gradient medium. The RBC-non-B-cell complexes with higher densities settle down through the medium, forming a pellet. The least dense plasma fraction forms the topmost layer. The less dense CLL cells form a white layer at the plasma-density medium interface.
Carefully transfer the white layer into a fresh tube. Centrifuge again to obtain a pellet containing purified CLL cells. In the following protocol, we show the isolation of chronic lymphocytic leukemia cells, from patient blood samples.
- Obtain peripheral blood samples from previously consented chronic lymphocytic leukemia patients in EDTA blood collection tubes accompanied by the white cell count.
- Human blood samples should be regarded as hazardous, as they potentially contain bloodborne viruses. Please ensure that these samples are handled in a Class II Biosafety Cabinet and that the user is wearing a lab-coat and gloves at all times. Dispose of blood contaminated materials and disinfect these.
- If the white cell count is equal or greater than 40 million cells per milliliter, dilute the sample at a ratio of 1 to 1 with RT CLL wash buffer. Then aliquot RT density gradient medium into an appropriately sized conical centrifuge tube for the sample. Carefully layer the sample on top of the medium and centrifuge at 400 times G for 30 minutes at RT.
Using a plastic pasteur pipette, gently harvest the white layer of mononuclear cells that collected at the interface of the density gradient medium and CLL wash buffer. Transfer this isolated monolayer into a fresh 50 milliliter conical centrifuge tube. To wash the cells, add 40 milliliters of CLL wash buffer to this monolayer and centrifuge at 300 times G for 10 minutes at room temperature.
Discard the supernatant, flick the bottom of the tube to re-suspend the pellet, and repeat this wash. Discard the supernatant, flick the bottom of the tube again, and then re-suspend the cell pellet in a set volume of CLL wash buffer, depending on the size of the pellet. After counting the cells and after flow cytometry to check the cell purity, place the cells into tissue culture plates at the required cell density, ready for stimulation, or drug treatment.

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1.7K Views. - In chronic lymphocytic leukemia, or CLL patients, B cell lymphocytes significantly increase in the bone marrow and blood. To isolate these CLL cells from a blood sample, first, treat the sample with a human B cell enrichment cocktail that separates B cells using negative selection. The antibodies in the cocktail specifically recognize and cross-link the antigens expressed by non B cells and erythrocytes, forming dense complexes. Next, layer the sample over a density gradient medium h...

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<table><tbody><tr><td>15 mL Tube&nbsp;</td><td>Griener Bio one</td><td>188271</td><td></td></tr><tr><td>1.5 mL microcentrifuge Tubes&nbsp;&nbsp;</td><td>Griener Bio one</td><td>616201</td><td></td></tr><tr><td>DMSO&nbsp;</td><td>Sigma</td><td>&nbsp;D2650</td><td></td></tr><tr><td>EDTA&nbsp;</td><td>Sigma&nbsp;</td><td>EDS</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher</td><td>10500064</td><td></td></tr><tr><td>Histopaque1077 density gradient media&nbsp;&nbsp;</td><td>Sigma</td><td>H8889</td><td></td></tr><tr><td>Trypan Blue Solution&nbsp;</td><td>Thermofisher</td><td>15250061</td><td></td></tr><tr><td>PBS Tablets&nbsp;</td><td>Fisher Scientific</td><td>BR0014G</td><td></td></tr><tr><td>RosetteSep Human B Cell Enrichment Cocktail&nbsp;</td><td>Stem Cell Technologies</td><td>15064</td><td></td></tr><tr><td>Sigma 3-16P&nbsp;&nbsp;</td><td>SciQuip</td><td></td><td>Centrifuge</td></tr></tbody></table>

Density Gradient Centrifugation: A Method to Isolate CLL Cells from Peripheral Blood

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Name	Company	Catalog Number	Comments
15 mL Tube	Griener Bio one	188271
1.5 mL microcentrifuge Tubes	Griener Bio one	616201
DMSO	Sigma	D2650
EDTA	Sigma	EDS
Fetal Bovine Serum	Thermofisher	10500064
Histopaque1077 density gradient media	Sigma	H8889
Trypan Blue Solution	Thermofisher	15250061
PBS Tablets	Fisher Scientific	BR0014G
RosetteSep Human B Cell Enrichment Cocktail	Stem Cell Technologies	15064
Sigma 3-16P	SciQuip		Centrifuge