Blood, plasma and serum collection

  • Blood is collected at 6-7.5, 12-13.5 and 18-19.5 months.
  • At each time point, for each strain, 16 female and 16 males are subjected to blood collection twice.
  • For every blood collection, we use 3 B6 males (8 to 12 weeks) as internal controls.
  • Before blood collection, scale mouse body weight.
  • Blood is collected by peri-orbital bleeding puncture.
  • * If mouse body weight is less than 15g, after the bleeding, IP 2ml saline to keep mice from dehydration.
  • Blood collection will follow four different protocols based on the different experimental objectives.
  • Mice are chipped after being used for Blood-1, 2 protocols.

Protocol 1: Blood-1

Objectives:

  1. For immune aging markers, measuring by FCS
  2. For chemistry analyses

Materials:

  1. For each sample we need to prepare: I) One microcontainer, 2 Eppendorf tubes. Label the tubes as MMDDYY-SampleID(1,2,3,…)-(A,B,C); II) 1 dilution vial, label with the sample ID(1,2,3…)
  2. In dilution vial, add 20ml isotonic diluent (Company, Cat#)
  3. FCS buffer (with 2% FBS)

Procedures:

  1. Blood is collected in tube-A (microcontainer). This protocol requires 270ul blood (about 13 drops, plus one capillary tube).
  2. Add 10ul of whole blood to each dilution vial, reverse the vial for several time to mix samples. Cell number will be counted by (Machine name and company).
  3. Separate plasma from Tube A by Centrifuging at 14,000rpm, 10 mins, 4C.
  4. Measure 140ul plasma and transfer into tube C.
  5. Measure the volume of left plasma and transfer into tube B. Record the volume.
  6. Save the plasma samples at –70C.
  7. Tap the tube A to loosen the pellet and then add 400ul of FCS buffer (with 2% FBS). Tap it again. Don’t vortex. Send samples for Immune aging marker tests.

Protocol 2: Blood-2

Objectives:

  1. Collect fresh whole blood for hematology assays.
  2. Collect plasma for further use.

Materials:

For each sample, preparing

  1. 2 microcontainers labeled with number;
  2. 1 Eppendorf 1.5ml tube. Label the tube as MMDDYY-SampleID(1,2,3,…).

Procedures:

  1. Blood is collected in hematology assay tube. Minimum required 270ul blood (about 11 drops plus one capillary tube). Keep blood samples at room temperature until analysis (up to 3 hours).
  2. Transfer 210ul whole blood to the second microcontainer and send samples for hematology assays.
  3. Plasma in the first container is separated by centrifuging at 14,000rpm, 10 mins, and 4C.
  4. Measure the volume of plasma and transfer to the labeled Eppendorf tube. Plasma is saved under –70C.

Protocol 3: Blood-3

Objective: Plasma for chemistry analyses

Materials:

For each sample we need to prepare 3 Eppendorf tubes. Label the tubes as MMDDYY-SampleID(1,2,3,…)-(A,B,C)

Procedures:

  1. Three weeks after the first blood collection, mice used in Protocol Blood-1 are subjected to the second blood collection.
  2. Blood is collected in 1.5ml Eppendorf tube-A (containing 7.5ul Heparin 1000u/ml). Minimum required 230ul blood (about 10 drops). Keep blood samples on ice.
  3. Centrifuge at 14,000 rpm, 5 mins, 4C.
  4. Transfer the supernatant to Eppendorf tube-A.
  5. Measure 110ul plasma and transfer to Eppendorf tube-B.
  6. Measure the volume of plasma that is left in the tube-B. Record the volume.
  7. Plasma samples are saved at –70C.

Protocol 4: Blood-4

Objective: Collect plasma for hormone and glucose assays.

Materials:

For each sample, preparing 1 microtainer, label as 1, 2, ….

Prepare 3 Eppendorf tubes. Label the tubes as MMDDYY-SampleID(1,2,3,…)-(A,B,C,).

One touch glucose measurement tools (machine and strips)

Procedures:

  1. Three weeks after the first blood collection, mice used in Protocol Blood-2 are subjected to the second blood collection.
  2. 7:30 a.m. take food off, fast mice for 4 hours to 11:30 a.m.
  3. Blood is collected in microtainer. Minimum required 250ul blood (about 13 drops).
  4. Before bleeding, measure the body weight.
  5. Use the One Touch to measure the glucose concentration
  6. Centrifuge at 14,000rpm, 10 mins, 4 deg C.
  7. Transfer the supernatant to Eppendorf tube-A (65ul for leptin and insulin assays); tube-B (30ul for IGF-1 assays).
  8. For insulin and leptin assays: measure 30ul plasma and transfer to Eppendorf tube-C.
  9. For IGF-1 assay: measure 25ul plasma and transfer to Eppendorf tub-D.
  10. Save Tube C and D in –70C.
  1. For glucose test: using the micro-glucose measuring machine to measure the plasma that is left in tube-B. Repeat three times for each samples. Record the results.
  2. Measure the volume of plasma that is left in Tube-B. Record the volume.
  3. Save Tube-B in –70C.

Protocol 5: Blood-5

1. We prepare three tubes for each sample. One microtainer, two eppendorf tubes. For Blood 5 protocol, we need to use the yellow microtainer.

2. Since we need 155 uL serum for the chemistry analyzer, we have to get at least 260 ul of blood (about 13 to 15 drops).