Sick mouse take down

Updated November 5th, 2021

Collect blood in EDTA coated tube either cheek bleed (on live mouse) or cardiac bleed (promptly after euthanasia) for CBC. Collect extra blood for Linage analysis.
Isolate both of hind limbs, spleen, or other tissues of interest (ex.Thymus).
Flush tibias and femurs with a 25 gauge needle and 5 mL Luer lock syringe. Suspend cells in 3 mL of staining media (magnesium/calcium free HBSS + 2% bovine serum). Pass through a 100 micron mesh filter. Count cells on a hemacytometer.
Weigh spleen using analytical scale. Cut half of the spleen for 10% formalin preservation for paraffin embedding / H & E staining. For splenocytes analysis, macerate other half of spleen or 50mg -60mg between two frosted slides in 3 ml of staining media. Use a 25 gauge needle and 5 mL Luer lock syringe to further break the clumps and pass through a 100 micron mesh filter. Count cells on a hemacytometer.
Cytospin: take 10ul of cells and dilute with 190ul staining media in a eppendorf tube. Prepare slides using Cytospin apparatus and use Cytospin machine at 1000 rpm for 5 minutes. Air dry the slide and stain using the HEMA 3 STAT Pack assay reagents and protocol.
Freeze down cells: 10 million bone marrow and/or spleen cells per mL in freezing media containing 90% FBS + 10% DMSO. Store in Mr. Frosty in -80˚C.
Lineage analysis for flow cytometry: stain bone marrow cells and spleen cells according to experiment.

8) Preserve RNA either by sorting leukemia cells or directly banking 100K cells in RLT or RNA-protect.