How many PBMCs does mouse blood have?

Typically, 200–300 μl of blood were obtained from each mouse, with the blood yielding approximately 3–5 X 106 PBMCs/ml, which is sufficient for the immunization of one mouse.

How do I get PBMC from mice?

My protocol to get PBMCs from mouse blood is a bit crude but simple. Transfer the blood to a vial with 9 ml distilled H2O and mix by inverting a couple of times (this will kill the red blood cells). Immediately after mixing add 1 ml of 10X HBSS or 10X PBS.

Can you culture PBMCs?

PBMCs can be cultured for 5-7 days in 24- or 96-well plates, using supplemented RPMI-1640 medium, and incubated at 37°C in a humidified, 5% CO2 atmosphere.

How long does PBMC last at 80?

1.5 years
Cryopreserved PBMCs have been reported to be stored up to 1.5 years, in a mechanical −70 °C/-80 °C freezer, depending on container type used, without effecting viable cell recovery [16].

How many T cells does a mouse have?

A typical mouse spleen weighs between 80 and 120 mg and contains up to 1×108 splenocytes. T cells constitute roughly 25% of all splenocytes, with the CD4+ T cells accounting for approximately two thirds of the total T cell population, and CD8+ T cells making up the remaining one third.

How do you get the serum from mouse blood?

To separate serum, allow blood in red top collection tubes (“Vacutainer”) to clot at room temperature, undisturbed for a minimum of 30 to a maximum of 60 minutes. 2. Remove clot by centrifugation at 1,000-1,500 x g for 10 minutes using a refrigerated centrifuge.

How do I freeze human PBMCs?

Place cryogenic vials immediately into an isopropanol freezing container (e.g. Nalgene® Mr. Frosty). Place the container in a -80°C freezer overnight. Note: Do not let cells sit in cryopreservation medium at room temperature.

Can you freeze PBMC?

Peripheral blood mononuclear cells (PBMC) can be frozen and stored in a cryoprotective media containing 10% dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS), then thawed rapidly. Cells frozen and thawed in this manner should have an acceptable yield and viability post-storage.

How do you store PBMC in liquid nitrogen?

Keep on ice and transfer rapidly. For long-term storage, transfer the vials of frozen PBMCs to vapor phase liquid nitrogen (below -135°C). Minimize exposure to room temperature by placing vials on dry ice during transfer from the -80°C freezer to liquid nitrogen. Note: Long-term storage at -80°C is not recommended.

How do you isolate B cells from a mouse spleen?

Aspirate off the supernatant, resuspend the cells in 1mL MACS buffer, and filter the cell suspension through a 70µm strainer into a 50mL conical tube. Rinse the tube and strainer. Transfer the filtrates to a 15mL conical tube and bring the final volume to 4mL. The cells are now ready for sorting.