Age+Associated+Stem+Cell+Autoimmunity+Experiment+11-23-12

11-23-12 Planning for experiment started.

Basically, I will take 1 old C57BL/6 mouse (mouse was born on _ which makes them _ old now), and 1 young C57BL/6 mouse (mouse was born on _ which makes them _ old now). I will then take the spleen and bone marrow from each mouse to obtain the splenocytes and stem cells from each mouse. Every combination of old and young splenocytes and stem cells will then be tested in a T cell assay (old splenocytes + young stem cells, young splenocytes + young stem cells, old splenocytes + old stem cells, young splenocytes old stem cells). The stem cells will be the cells that are labeled with chromium in the killing assay. This experiment will demonstrate whether or not old splenocytes attack young stem cells more often than young splenocytes attack young stem cells.

Version of Chromium Release Assay Protocol for Age Associated Stem Cell Autoimmunity Experiment 11-23-12

Some Questions in preparation for the experiment Do I have enough chromium? -I'll need 200 uL 1mCi/mL Cr for each incubation, and I have 2 incubations so I will need 400 uL 1 mCi/mL Cr. I currently have 260 uL of 1 mCi/mL Cr. Therefore, I could use 130 uL instead of 200 uL per incubation (1 incubation for old stem cells and 1 incubation for young stem cells). I think this should work. I have labelled cells that don't label extremely (B16F10 cells) well with 100 uL chromium, and the cells still labeled well. I was able to obtain counts at 1000 cpm or higher.

Note: The previous 100 uL cell labeling experiment can be found here: Chromium Release Test 10-18-12

Do I have enough reagents in the stem cell kit? Yes. I basically have a full kit. One round of use of the kit can isolate up to 8e8 cells. The kit has no expiration date on it.

Do I expect to obtain enough cells for the experiment? -I should definitely have enough splenocytes. I only need about 9.5 million cells per spleen, and I should get about 100 million cells per spleen. -I should have enough stem cells. I only need 4e5 cells per bone marrow, and the last time I isolated stem cells I obtained about 1.4e6 cells.

Note: The previous stem cell isolation experiment can be found here: Practice isolating stem cells from mouse 6-5-12

Actual experiment carried out on 1-4-13 notes in labnotebook found on pg 122-124 of pdf found here "F:\kurt\storage\CIM Research Folder\kwhittem\Records in CIM Folder\Scans of Notebooks\lab notebook\6 8-4-12 through 4-9-13\Notebook 8-4-12 to 4-9-13.pdf"

Aged Mouse from this group: Old C57BL s 6 mice received by Shen portal 11-8-12 Aged Mouse: 1 yr 11 months 25 days

Young Mouse from this group: 11-20-12 received young C57BL s 6 mice Young Mouse: 3 months 11 days

Stem Cells: hematopoietic progenitor cells isolated by negative selection by removing CD5, CD11b, CD19, CD45R, Ly-6G/C, TER119, and 7-4 positive cells with the EasySep Negative Selection Mouse Hematopoietic progenitor Cell Enrichment Kit (Cat 19756)

Splenocytes: all cells from spleen.

See summary of experiment here "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\Summary aged-young cr release assay 1-5-13.docx"

Graph of young stem cell e_t ratio of 3 "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\young stem cell e_t ratio of 3 6-26-13.pzf" "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\p-values for e_t ratio of 3.xlsx" "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\young stem cell e_t ratio of 3 6-26-13.svg" ^renamed to !^ "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\Figure 1a.svg" "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\Figure 1a.pdf"

Graph of old stem cell e_t ratio of 3 "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\old stem cells e_t ratio of 3 6-26-13.pzf" "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\p-values for e_t ratio of 3.xlsx" "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\old stem cells e_t ratio of 3 6-26-13.svg" ^renamed to !^ "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\Figure 1b.svg" "F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay\Figure 1b.pdf"

See F:\kurt\storage\CIM Research Folder\DR\2013\1-5-13\old-young cr release assay for supporting files

Stored Old Mouse Serum 1-4-13 at Kurt Mice 2 7-15-10 box at -20 C Stored Young Mouse Serum 1-4-13 at Kurt Mice 2 7-15-10 box at -20 C

Used 120 uL Cr51 from 2012 099 which was received 9-5-12 Used 80 uL Cr51 from 2012 113 which was received 10-17-12

There is 70 uL left in 2012 113. 100 uL was used per sample.

I obtained 4.5e5 stem cells from a young mouse I obtained 7.5e5 stem cells from an old mouse

Old splenocytes: 4.5e7 cells Young splenocytes: 8.63e7 cells

1-9-13

notes in labnotebook found on pg 125-128 of pdf found here "F:\kurt\storage\CIM Research Folder\kwhittem\Records in CIM Folder\Scans of Notebooks\lab notebook\6 8-4-12 through 4-9-13\Notebook 8-4-12 to 4-9-13.pdf"

Two of the old mice were wounded and the caretakers asked me if one of them should be placed onto an anti-itch cream treatment. I terminated both mice instead. Blood, splenocytes, and bone marrow cells were collected and frozen. The radioactive sodium chromate came later that day as well. I have one old mouse left which could be dying very soon. Therefore, I have decided to terminate the last old mouse as well as the other four young mice, and perform the killing assay.

This time when I do the experiment I will avoid pipetting small volumes such as 3 uL with the 3:1 Sp:St condition. Instead, I will make a high dilution and use a larger volume to place into the well. Also, this time I will not isolate the stem cells. I will just use all of the bone marrow cells just as I used all of the splenocytes rather than isolating the T cells. Additionally, I will combine cells from different individuals of the same age group to rule out the possibility of reactions which are merely against cells from a different individual.

I will test many conditions. I have 3 aged mice. A1 non-wounded A2 1 wound A3 2 large wounds

Conditions splenocytes + stem cells Y1+A1 Y1+A2 Y1+A3 Y1+Y2 Y2+Y1 Y1+Y1 Y2+Y2 Y3+A1 Y3+Y3

A1+Y1 A1+Y2 A1+Y3 A2+Y1 A2+Y2 A3+Y1 A3+Y2 A1+A1 A1+A2 A1+A3 A2+A1 A2+A2 A2+A3 A3+A1 A3+A2 A3+A3

Plate maps for this experiment found here F:\kurt\storage\CIM Research Folder\DR\2013\1-10-13\CTL\plate_maps

Chromium release assay experiment performed 1-10-13 (Thursday)

Images of the counts on the plates found here F:\kurt\storage\CIM Research Folder\DR\2013\1-14-13\CTL\Plate Images

Data for experiment "F:\kurt\storage\CIM Research Folder\DR\2013\1-18-13\CTL\CTL 2 1-18-13.xlsx"

Summary of Experiment "F:\kurt\storage\CIM Research Folder\DR\2013\1-18-13\CTL\Aged Young Cr Release Assay 1-22-13.pptx"

Graphpad prism files F:\kurt\storage\CIM Research Folder\DR\2013\1-18-13\CTL

some paragraphs about experiment { I hypothesized that part of the aging process is due to an autoimmune reaction against stem cells necessary for repairing damaged tissue. The prevalence of autoimmune diseases in general increases with age, and the prevalence of cancer also increases with age. Cancer cells share some similarities with stem cells which are needed to repair damaged tissue. The immune system may get confused with age and mistake stem cells for cancer cells resulting in an age associated stem cell autoimmunity. One possible experiment to test this hypothesis involves performing a cytotoxic lymphocyte assay (CTL assay). I hypothesized that splenocytes form aged donors target young stem cells more than splenocytes from young donors. The experiments performed support this hypothesis. Some additional unintended observations were also made.

In the CTL experiment there were 3 young mice and 3 aged mice. However, in the aged mouse group mouse 1 had no visible wounds, mouse 2 had 1 wound on its side, and mouse 3 had 2 wounds (one large wound on side and very large wound on back of neck). The cause of these wounds is unknown. Splenocytes and stem cells were collected from all six mice and combined in every possible combination. Figure 1 addresses the question: How do aged and young splenocytes compare in regards to their reactivity for young stem cells? Figure 2 addresses the question: How do aged and young splenocytes compare in regards to their reactivity for aged stem cells? Figure 3 addresses the question: How do aged splenocytes from an unwounded mouse (1), a mouse with 1 wound (2), and a mouse with two large wounds (3) compare in regards to their reactivity for aged splenocytes?

}