Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

In this video protocol, you'll see how to dissociate mouse spleens using Gentle Max, an automated benchtop device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation spleens are filtered with Max Pres separation filters, centrifuge, and suspended for further applications. Hi, my name is Melanie mlu.

I'm a scientist at Milton Biotech here in BE Lab Park, Germany. Today I will show you the preparation of a single cell suspension out of mouse spleen by using this GenX dissociation. The GenX dissociation allows the very easy and fast preparation of a single cell suspension.

So let's get started. To avoid tedious and often painful manual dissociations, the gentle max di associator allows one to dissociate tissue very efficiently under controlled and reproducible conditions. Tissue is gently dissociated using violet C tubes into viable single cell suspensions.

The C tube provides a septum sealed opening into the center of the cap, allowing one to pipette from the tube while always maintaining sterility. To suspend the dissociated cells, you will need to use the proper P-B-S-B-S-A-E-D-T-A buffer. The buffer is prepared by adding one part of BSA stock solution to 20 parts of AutoMax fencing solution containing EDTA.

Using the Max Pres separation filters, dissociate tissue samples are freed from all cell clumps promoting successful max separation and subsequent analysis. To begin the procedure, we load the C tube with a volume of buffer. According to the mass of the spleens, a healthy spleen of a six to seven week old female biopsy mouse weighs about 80 to 120 milligrams.

Every one to two spleens of this mass requires three milliliters of buffer. Do not exceed a total volume of 10 milliliters. We can now add our dissected spleen to the C tube containing three milliliters of buffer Spleens from older animals or animals with infections are often larger and must be weighed in buffer.

The buffer volumes must then be scaled accordingly, but again may not exceed a total volume of 10 milliliters. Now, make sure that the C tube cap is tight and attach the C tube to the dissociation With a cap down, power up the dissociation for one to two spleens select Program M spleen oh one. The program will take 56 seconds to complete if the number of spleens is greater use program M spleen oh four.

This program can be used for up to 720 milligrams of spleen tissue. After completion of the program, the native spleen tissue is dissociated into a PLE cyte suspension, which looks like this centrifuge briefly to collect the sample at the bottom of the tube. This will ensure that the rotor and stator are freed of all cells which are collected at the bottom of the C tube.

We remove the sample through the septum sealed opening in the C tube cap using a thousand microliter pipetter. To begin the filtration step, we apply the dissociated tissue to the pres separation filter or a strainer with 30 micron pore size. The strainer should fit a 15 milliliter tube for one to two spleens or a 50 milliliter tube for more than two spleens.

Let the cell suspension pass through, then wash the filter by applying five milliliters of buffer. Now centrifuge the tube with the cell suspension at 300 Gs at room temperature for 10 minutes to collect all cytes in a spleen cell pellet. Now that we have a pellet of dissociated cytes, we must aspirate the super natin and resuspend the cell pellet in an appropriate amount of buffer spleen dissociation.

Using the gentle max will yield a high percentage of viable PLE cytes, which is demonstrated by flow cytometry using the max quant. Dead cells are stained by Propidium iodide and the example provided. The forward side scattered dot plot shows a prepared SP cytes.Okay.

I've shown you how to dissociate the spleen tissue by using this gentle X dissociation. When you do this procedure, it's important to adjust the amount of buffer to the sample size and to choose the right Gentle X program. Okay, that's it.

Good luck with your experiment.