Aug 30, 2022
SPARC - Preparation of Plasma Samples from Rats
- J Paul Robinson1
- 1Purdue University
- SPARCTech. support email: [email protected]
Protocol Citation: J Paul Robinson 2022. SPARC - Preparation of Plasma Samples from Rats. protocols.io https://dx.doi.org/10.17504/protocols.io.261geodpol47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 17, 2019
Last Modified: August 30, 2022
Protocol Integer ID: 31106
Keywords: rat plasma collection, plasma from live rat experiment, preparation of plasma sample, further hormone assay, plasma sample, live rat experiment, plasma, sparc, preparation
Abstract
Objective: To collect plasma from live rat experiments for storage at -20˚C for further hormone assays.
Protocol materials
Protease Inbibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #P2714-1BTL
Troubleshooting
Turn on the Hettich Mikro 22R refrigerated centrifuge so it can begin to cool down to 4 °C .
Hettich Mikro 22R refrigerated centrifuge (Cat # 1110, https://www.hettweb.com/)
Equipment
Hettich Mikro 22R refrigerated centrifuge
NAME
Centrifuge
TYPE
Hettich
BRAND
Cat # 1110
SKU
LINK
Add 15 µL of Protease Inhibitor Cocktail to each of 5 Culex tubes (Vial Clear 8mm Crimp Rnd Bottom
300 µl Lot:0000071223 MicroLiter Wheaton Company)
Protease Inbibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #P2714-1BTL
Firmly place cap (Snap Cap 8mm, Natural with Cut T/S Septa Lot 0000000308 MicroLiter Wheaton Company) making sure it is on good and level. (these are plastic reusable labels from BASI inc)
Place the plastic numbers (re-usable-do not dispose of these number labels)1-5 one on each Culex tube, place up from bottom of tube.
Equipment
Culex Automated Blood Collection System
NAME
Blood Collection
TYPE
Culex
BRAND
NxT
SKU
LINK
Place the Culex tubes on the Culex machine in slots 1-5.
Put the labels on the strip tubes if they are not already.
Equipment
Labels
NAME
Dymo
BRAND
Cat# 1750283
SKU
LINK
When the first sample comes off the Culex, centrifuge on program #3 (4˚C, 10 minutes, 1000 x g (3.000 rpm )1000 x g immediately.
Using the P100 pipette set at 85 µL l carefully try to remove as much of the plasma as possible and transfer to the appropriate pcr strip tube. If needed set to 15 µL and remove remaining plasma without disturbing the red cell layer.
Equipment
new equipment
NAME
Pipettman
BRAND
Cat#75874-574
SKU
LINK
If needed switch to the P100 set to 15 µL and remove remaining plasma without disturbing the red cell layer.
Keep the strip tubes in the freezer block (ISO Freeze) during the entire collection process.
Equipment
IsoFREEZE PCR Tube Chiller Rack
NAME
Freezer container
TYPE
RPI
BRAND
248002
SKU
LINK
Do this for each sample as it comes off the Culex machine, S1, S2, S3, S4, S5.
Place an orange dot on the label on top of all the tubes that have been collected using the orange sharpie. This just indicates the original tube from which the aliquots were taken.
Once the fifth sample has been collected, change the rotor on the Hettich Mikro 22R centrifuge to the one for strip tubes.
Change the program to program #2, which is 6.000 rpm (RCR 4020)10 minutes, 4˚C.
You will need to close the lid and press start and wait. It will recognize the rotor has been changed and will automatically STOP.
Once it is stopped open the lid again. Then close the lid and press START. It should now run through the program.
When stopped, remove the strip tube.
Using the P200 set to 25 µL and the P100 and make two aliquots of 25 µL for every sample. If any aliquot is short, write an “S” on the cap of that aliquot.
Once all samples have been aliquoted, spin them again on program #2 to get all the sample to the bottom.
25 µL samples must be transported using the ISO Freeze sample rack to keep temperature at 4 °C
Make sure all lids are on tight, and place samples in -20 °C freezer. Samples are added over time to the next plate to be run and placed in a labeled Ziploc bag in the -20 °C freezer.
Samples must then be logged into the next plate map using the MPLEX software. Print a copy after each sample set has been added to the plate map for recording purposes. The purpose of adding each sample to the Plate Map for the MPLEX software is to maintain a fully digitized record for each assay.