Chronologic List of Experiments


The following is the chronological log of experiments performed by David Knowles as a post doctoral fellow in the laboratory of Mohandas Narla.

Images were collected on a PC (on now obsolete SYQUEST disks which were backed up onto 1GB JAZ disks) and transferred to a SUN Workstation DataBase (accessible from both curlcurl.lbl.gov & bondi.lbl.gov) for analysis. Only the highest quality images were transferred and analyzed. Images were named as a numeric series appending the current date (eg the first image saved on march 5th would be mr501.tif. Note this is the first series of images in the list which is marked as being transferred.). The images, which are accessible to all users, are in the directory /home/curlcurl/davidk/Images/ and catalogued under several subdirectories which are named by the type of red cell (eg:norm - Normal red blood cells, see Legend below). Below these directories the images are catalogued by type of fluorescent label.

For Example: the first transferred entry below reads:
Mar5th95 * f-10f7 55ug/ml DK rbc   \f-10f7\mr5  1-50    (good images)

It tells you 50 images mr5,1-50 were collected of f-10f7 labeled at 55ug/ml on DK's rbcs, into the subdirectory /f-10f7/
The quickest way to find these images is to "cd /home/curlcurl/davidk/Images/" and to "find . | grep mr5" (standard UNIX search command) which out puts a list of the 50 images. For example, "./norm/f-10f7/mr5001.tif" tells you the image is of normal red blood cell labeled with f-10f7.

".dat" files are the analysis output a given file series and are readable. (See http://white.lbl.gov/~dwknowles/software/ )
For Example: the following entry tells you Control (Rh_Null) EMA images oc18,41-80 were collected into /ema/.
Oct18th97   Control (Rh_Null) EMA   \ema\ oc18 41-80
"find . | grep oc18" shows you that not only were those images stored in /norm/ema/images/ but also that day images 0c18,1-40 were stored into /Rh_null/ema/.
It also shows you the analysis output for images oc18,41-80 is in /norm/ema/ and has the name oc18-41-80.dat. "more oc18-41-80.dat" shows you the file.
The first 3 lines are printed below and tell you that this output was created by FIM_ANALYSIS version Vs.Fe97, followed by a header line and that the first
image in the series, oc18041 has a projection length "L(um)" of 4.34um a pipette radius "Rp(um)" of 0.46um an entrance density "Pe" of 1.85, cap density "Pc"
of 0.84 etc (see Software)
Vs.Fe97
File       L(um) Rp(um)   Pe   Pc Pmid L2(um)  L/Rp Pe/Pc G_AV  G_1  G_2 Pipd Entr Fit1 Midfit Fit2 Cap  Bi
oc18041     4.34   0.46 1.85 0.84 0.00   0.00  9.43  2.19 0.11 0.00 0.00    7   85   95      0  118 118   0

David Knowles



LEGEND


norm - Normal red blood cells
SAO - South Asian Ovalocytes
MiV - Miltenberger V
MiVctrl - Miltenberger V Control = Normal RBC from 77K
Marrow - normal rbc's from human bone marrow
R1 - reticulocytes from human bone marrow
4.1d - rbc's with 100% deficiency of protein 4.1
Hema -  Haemaglobin density fractionated normal RBC's
frag - Fragmentation images- cap vesiculation images

ema - eosin malamide 734g/mole, e=100,000, Mol.Probes
fma - fluorescein-5-maleimide. Mol.Probes
etsc - eosin-5-thiosemicarbazide. 737g/mole e=90,000. Mol.Probes
ftsc - fluorescein-5-thiosemicarbazide. Mol.Probes
flph - fluorescein phalloidin. e=75,000. 495,520nm Mol.Probes F-432
rhph - rhodamine phalloidin. e=75,000. 550,575nm Mol.Probes R-415

Brac18 - monoclonal anti band3
R10, 9A3, B14 - monoclonal anti glycophorin A
Bric10 (B10) - monoclonal anti glycophorin C
CHIP - fluorescine anti RBC water channel protein AQP1
dids - 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, disodium salt (DIDS)
f-dhpe - fluorescine PE
f-a-cd59 - fluorescine anti CD59

Anky Frag - 43kD Ankyrin Fragmant to Band3
f-anky - fluorescine conjugated 43kD Ankyrin Fragmant to (cytoplasmic) Band3

Magic Sol - 1mMNaN3 (3.3mg/50ml) 5mM Trolox (62mg/50ml)

* - Transferred to the SUN Workstation Station for analysis



Date  Sun Experiment    Image Series    Comment

DISK I

Feb3rd95  ema     feb03 1-25 chymo ?? times
Feb4th95  ema     feb04 1-35
Feb6th95  ema     feb06 1-19
Feb7th95  a-fl-pe     feb07 1-3
   c12-bodipy-pe    feb07 4-?
Feb9th95  c12 bodipy pe    feb9 1-13 Some parasite infected rbc's
Feb10th95  c5-bodipy pe    feb10 5-18 ??
Feb17th95  c12 bodipy pc    feb17 1-51
Feb21st95  c12 bodipy pe    feb21 1-49
Feb22nd95  c12 nbd pc    feb22 21-24
   c12 nbd ps    feb22 30-49
Feb26th95  c12 nbd ps    feb26 2-11
Feb28th95  rhph     feb28 1-40

Mar2nd95  n-fl-pe Avanti egg   mar02 1-10
   c12 nbd pc    mar02 34-39
   c12 nbd ps    mar02 41-52
Mar5th95 * f-10f7 55ug/ml DK rbc   \f-10f7\mr5  1-50    (good images)
   f-10f7     mar05 37-42
   f-10f7 Binding Isotherm!!  ??
Mar7th95  rhph (5uM)    mar07 14-20
   rhph (2.5uM)    mar07 21-40
Mar10th95 * ftsc     \ftsc\mar10 1-20 (1-10, pip 15-20) (nice images)
  * ftsc R10 14ug/ml)   \ftsc\mar10 21-30, 31-37 (pip)
  * ftsc R10 56ug/ml   \ftsc\mar10 41-47
Mar12th95  F-10f7 18ug/ml   mar12 1-15
   F-10f7 30ug/ml   mar12 47-49
Mar13th95  ftsc     mr1301 - 20
   ftsc + R10 70ug/ml   mr1321 - 26
Mar15th95 * ftsc + R10 70ug/ml   \ftsc\mr1501 - 30
  * ftsc     \ftsc\mr1531 - 50
   ema     mr1551 - 55
Mar16th95  ema (day old)    mr1601 - 20
   ema      mr1621 - 45
   ema + Brac18 50ug/ml   mr1646 - 55
   annexinV Malaria RBC   \annex\mar16 3-48
Mar20th95  ema + Brac18 5ug/ml   mr2001 - 23

Mar21st95  F-10f7 150ug/ml (Vesiculation)  \f-10f7\mr2101 - 19   Images 14-19 are the same cell
        mr2120 - 30 (310+-76/800ms)
   F-10f7 75ug/ml   mr2131 - 40 (436+-60/800ms)
   F-10f7 20ug/ml   mr2141 - 50 (156+-102/800ms)
Mar22nd95  F-10f7 100ug/ml   mr2201 -10 (414+-60c/800ms)
   F-10f7 50ug/ml   ?? mr2211 - 20 (335+-70/800ms)
   F-10f7 10ug/ml   ?? mr2221 - 30 (115+-6/800ms)
Mar23rd95  F-10f7 125ug/ml   ?? mr2301 - 10 (445+-42/800ms)
   F-10f7 35ug/ml   ?? mr2311 - 20 (240+-18/800ms)

Apr13th95  F-9A3 FAB 500ug/ml, Mohan RBC
   15-18 Same Cell Vesiculation  ap1301 - 41
Apr14th95  F-9A3 500ug/ml   ap1401 - 40 (Ves)
Apr20th95  F-9A3 Isotherm
   50ug/ml    ?? (26+-7/800ms)
   100ug/ml    ?? (34+-6/800ms)
   200ug/ml    ?? (43+-7/800ms)
   400ug/ml    ?? (63+-6/800ms)
   25ug/ml    ?? (21+-4/800ms)
   0ug/ml     ?? -2+-2/800ms

Apr21st95  F-9A3 25ug/ml    ?? (9+-2/800ms)
   F-9A3 100ug/ml   ap2121 - 25
   ema SAO    d:\oval\ap2126 - 33
   ema RBC    ap2134 - 40
Apr22nd95  ema, RBC receales   ap2201 - 15
   ema SAO, reseales   ap2216 - 46, ap2247 - 66
   ema SAO    ap2241 - 46
Apr23rd95  ema SAO    \oval\ap2301 - 40
        41 - 60 Ultra narrow pipette
   ema     ap2361 - 80 Ultra narrow pipette
   ema + R10 70ug/ml   d:\r10\ap2381 - 100
Apr24th95  rhph SAO    \oval\ap2421 -40
   rhph RBC    \oval\ap2441 - 60
        61 - 72 Ultra narrow pipette
Apr26th95  SAO rhph    ap2601 - 16

DISK II

Apr27th95  PE ?     \pe\ap27 1-11
Apr28th95  PE ?     \pe\ap28 1-20
   Ankyrin frag 85ug/ml   ap28 21 - 25 (see notes)

May12th95  ema     may12 1-13
May13th95  ema     may13 1-20
May16th95  nbd ps     \pe\may16 22-23    More on Disk 1(Frag)
May17th  n-f-pe +- ani FITC fab   \pe\may17 2-16??    (lousy images)
   n-f-pe + anti-Fl-texasRed  \pe\may17 17-57 ??    Capping (time seq) & Frag
May18th95  rhph     \rhph\may18 1-13
   rhph R10 20ug/ml   \rhph\may18 14-23
   rhph R10 80ug/ml   \rhph\may18 24-32
   rhph n-fl-pe    \rhph\may18 33-35 ??36-40, 42-80  (Frag, white)
   ftsc R10 63ug/ml   may18 81-88?
May18th95  ftsc     may19 1-32
   ??ftsc     may19 61-78
May19th95  ftsc     \ftsc\may19 1-9, 11, 22-29,31-33,(??51-60) 61-78 From Pre-Scil Analysis
   ftsc + R10 ??20ug/ml   \ftsc\may19 14,16-21,41-50  (Frag, nice)
   actual files    \ftsc\may19 1-9,11,14,16-27,29,31-33,35,
        41,44,46,50,52-60,71-72,74-78,80-82,84-86,88
May26th95  lipid?     may26 1-27     Frag
My31st95  SAO ema 100x lens   my310 4 - 10
Jn1st95  ema     d:\ema\jn01 1 - 20
   SAO ghosts + Anky Frag 120ul  \oval\jn01 21-40
Jn2nd95  SAO ema    \oval\jn02 1 - 20
   SAO ema Anky Frag 80ul  \oval\jn02 21 - 49
   SAO ema resaealed control  \oval\jn02 51 - 75
jun3rd95  ?? non red cella ??   \pe\jun03 2-36
Jn4th95  ema Norm Anky 5ul   \anky\jn04 1 - 20
Jn5th95  ema RBC Anky 5ul   \anky\jn05 1 - 20
  * MiV ftsc    \miv\jn05 21 - 49    Frag
  * MiVctrl ftsc    \miv\jn05 51 - 83
Jun6th95 * MiV ftsc    \ftsc\jun06 1-17
  * MiV ftsc R10 20ug/ml   \ftsc\jun06 21-34    (Frag)
   MiVctrl ftsc R10 20ug/ml  \miv\jn06 41-45
  * MiVctrl ftsc R10 20ug/ml  \miv\jn06 61 - 80
  * MiV ftsc R10 80ug/ml   \miv\jun06 81-107    (Frag)
  * MiVctrl ftsc R10 80ug/ml  \miv\jun06 111-119   (Fragmentation Seqence)
  * MiVctrl ftsc R10 80ug/ml  \miv\jun06 131-150   (Frag)
Jun8th95 * MiV ftsc    \miv\jun08 1-38    (Frag)
  * MiV fstc + R10 20ug/ml   \miv\jun08 41-57    (Frag)
  * ctrl ftsc     \miv\jun08 61-70
  * MiVctrl ema     \miv\jn08 71-90
  * MiV ema    \miv\jn08 91 -110
Jn9th95 * MiVctrl ema R10 40ug/ml  \miv\jn09 1 - 20
  * MiV ema R10 40ug/ml   \miv\jn09 21 - 40    (35,36 Frag)
Jn12th95 * MiV ema    \miv\jn12 1 - 20
  * MiVctrl ema R10 40ug/ml  \miv\jn12 21 - 40
Jn13th95 * MiV ema R10 40ug/ml   jn13 1 40
  * MiVctrl ema R10 40ug/ml  jn13 41-47, 48 - 50(pip)
Jn16th95 * R10 ema RBC    \r10\jn16 1 - 20
 

Jn21st95  ema Brac18 20ug/ml   jn21 1 - 40
   ema     jn21 41 - 80
Jn24th95 * etsc magic    \etsc\jn24 1 - 40
   etsc Brac18 20ug/ml magic  \etscb18\jn24 41 - 92, 93 - 100(pip)
Jy1st95  ema Brac18 2ug/ml   \brac18\jy1 1 - 33, 34 -40(pip)
Jy5th95  etsc Brac18 2ug/ml   \brac18\jy5 1 - 40
   ema Brac18 2ug/ml   \brac18\jy5 41 - 60

Jy6th95  etsc Brac18 0.2ug/ml   \brac18\jy6 1 - 40
   ema Brac18 0.2ug/ml   \brac18\jy6 41 - 80

Sep7th95  ema     se07 1 - 40
Sep8th95  ema Brac18 2ug/ml   \brac18\se08 1 - 40
   ema Brac18 20ug/ml   \brac18\se08 41 - 80
Sep11th95  ema Brac18 20ug/ml   \brac18\se11 1 - 40
   ema Brac18 0.2ug/ml   \brac18\se11 41 - 80
Sep12th95  ema Brac18 2ug/ml   \brac18\se12 1 - 40
   ema Brac18 0.2ug/ml   \brac18\se12 41 - 80

Sep15th95 * etsc     \etsc\se15 1 - 40
   etsc Brac18 20ug/ml   \etscb18\se15 41 - 80
   etsc Brac18 2ug/ml   \etscb18\se15 81 - 100

Sep18th95  ema Brac18 20ug/ml   \brac18\se18 01 - 17
Sep19th95  ema Brac18 20ug/ml   \brac18\se19 01 - 20
   etsc Brac18 20ug/ml   \etscb18\se19 21 - 40
Sep26th95  ema Brac18 20ug/ml   \brac18\se26 01 - 40
Sep27th95  ema Brac18 2ug/ml   \brac18\se27 01 - 60
   ema Brac18 0.2ug/ml   \brac18\se27 61 - 100
Sep29th95 * etsc     \etsc\se29 01 - 40
   etsc Brac18 20ug/ml   \etscb18\se29 41 - 80
   etsc Brac18 2ug/ml   \etscb18\se29 81 - 120
Oct2nd95  etsc Brac18 0.2ug/ml   oc02 01 - 40

Oct3rd95  rhph     \rhph\oc03 01 - 60
   rhph Brac18 20ug/ml   ?? oc0361 -

Oct27th95 * CHIP (dil 2ul + 8ul)   oc27 01 - 40 (see notes for times)
Oct30th95 * CHIP O+(Aura RBC)   oc30 01 - 20 (+ times on a few points)
Nov1st95  CHIP Coulten(a-b-)   nv01 01 - 7 (no binding)
   CHIP Co(a+b+)    nv01 08 - 16 (binding)
Nov2nd95  CHIP Co(a-b-)    nv02 01 - 10
   CHIP Co(a+b+)    nv02 11 - 20
Nov3rd95 * CHIP O+ (aura)    nv03 01 - 40
Nov4th95 * CHIP O+    nv04 01 - 60
   CHIP O+ Brac18 20ug/ml  nv04 61 - 100 (mu's done)

Jan11th96  flph     \fl-ph\jan11 50 - 54
   rhph     \rh-ph\jan11 55 -64
Jan12th96  flph     \fl-ph\jan12 01 - 14
Jan18th96  rhph (reanalysis??)   \rh-ph\feb28 01 - 40
        \rh-ph\mar07 01 -40
        \rh-ph\mr30 01 - 60

??   reanalysis    \rh-ph\may18 01 - 80 Lipid >59 (Leann??!)
        \rh-ph\oc03 01 - 60
Feb29th96  rhph     \rh-ph\feb29 01 - ??

Mar11th96  Marrow F-Brac18 FAB 800ug/ml \marrow\mr1101 - 50
Mar12th96  Marrow ema    \marrow\mr1201 - 20 normal
   R1 ema    \marrow\mr1221 - 30
Mar13th96  R1 ema    \marrow\mr1301 - 20
   R1 F-Brac18 FAB   \marrow\mr1321 - 40
Mar20th96  F-Brac18 FAB Off Rate   \marrow\
   100ug/ml    mr2001 - 10
   400ug/ml    mr2011 - 20
   800ug/ml    mr2021 - 30
   200ug/ml    mr2031 - 44
   100ug/ml    mr2045 - 55 (times included)

Mar27th96  F-Bric10 100ug/ml   mr2801 - 20
   F-Bric10 10ug/ml   mr2821 - 30
   F-Bric10 50ug/ml   \b10\mr2831 - 50
   F-B10 5ug/ml    \b10\mr2851 -60
   F-B10 2ug/ml    \b10\mr2861 - 70
   F-Bric10 1ug/ml   \b10\mr2871 - 80
   F-Bric10 0.2ugml   \b10\ap0101 - 10
   F-B10 0.1ug/ml   \b10\ap0111 - 20

   F-Bric10 Isotherm
   100ug/ml    85+-4.2/1000ms
   10ug/ml    52+-5.4/1000ms
   5ug/ml     64+-6.4/1000ms
   50ug/ml    81+-10.6/1000ms
   1ug/ml     66+-6/1000ms
   2ug/ml     80+-6/1000ms
   0.2ug/ml    79+-21/1000ms
   0.1ug/ml    30+-10/1000ms

Apr2nd96  F-Bric10 5ug/ml + R10 70 ug/ml \b10\ap0201 - 30

Apr10th96  4.1d F-B10 25ug/ml   \4.1d\b10\ap1001 - 50
   4.1d B10 25ug/ml ema   \4.1d\b10\ema\ap1051 -80
Apr11th96  4.1d B10 etsc    \4.1d\b10\etsc\ap1101 - 20
Apr12th96  4.1d B10 etsc    \4.1d\b10\etsc\ap1201 - 30
   4.1d B10 ema    \4.1d\b10\ema\ap1231 - 50 (times inc)
May20th96  Marrow & R1 F-DHPE   \f-dhpe\my2001 - 30
May21st96  Marrow & R1 F-DHPE   \f-dhpe\mr2101 - 10
May22nd96  Marrow & R1 F-DHPE   \f-dhpe\my2201 - 25

May24th96  rhph     \rhph\my2401 - 20
   Marrow rhph (R1's dont label)  \marrow\rh-ph\my2421 - 30
   Marrow rhph F-DHPE   \marrow\rhph\f-dhpe\my2431 - 40
   R1 F-DHPE    \marrow\f-dhpe\my2441 - 50
Jun10th96  Marrow F-DHPE   \marrow\f-dhpe\jn10 ??
Jun11th96  Marrow F-DHPE (flu and white)  \marrow\f-dhpe\jn1101 - 20
Jun12th96  Marrow F-DHPE (flu and white)  \marrow\f-dhpe\jn1201 - 10
Jun13th96  Marrow & R1 F-B10   \marrow\f-b10\jn1001 - 40
Jun19th96  Marrow ema    \marrow\ema\jn1901 - 13
Jun21st96  Marrow  & R1 ema   \marrow\ema\jn21 1 _ ??

Jul28th96  ema     \ema\jy2801 - 10
Jul29th96  ema     \ema\jy29 1-20, 21-50(pip)
   ema ghost    \ema\gho\jy29 51 - 80
Jy30th96  ema ghost    \ema\gho\jy30 1-31

Aug5th96  rhph + fma (odd fma, even rhph)\rhph&fma\au05 1-60, 61-70(pip)
Aug15th96  rhph + fma    \rhph&fma\au15 1-40
   rhph + fma + Brac18 50ug/ml  \rhph&fma\brac18\au15 41-64
Aug16th96  rhph fma Brac18 50ug/ml  \rhph&fma\brac18\au16 1 -40
Aug19th96  rhph fma Brac18 50ug/ml  \rhph&fma\brac18\au19 1-38, 39-60
Aug21st96  rhph fma Brac18 50ug/ml  \rhph&fma\brac18\au21 1-60

Sep16th96  F-Brac18 Isotherm   \f-b18\
   100ug/ml (no magic)   se16 1-10 (354+-76/1000ms)
   100ug/ml (magic)   se17 1-10 (183+-17/1000ms)
   100ug/ml magic, lamp adj  se17 11-20 (239+-31/1000ms)
   100ug/ml deflate c PBS no mag se17 21-30 (414+-34/1000ms)!!

   100ug/ml no magic (1:500)  se17 41-50 (549+-29/1000ms)
   50ug/ml    se17 51-60 (409+-44/1000ms)
   20ug/ml    se17 61-70 (375+-50/1000ms)
   10ug/ml    se17 71-80 (473+-38/1000ms)
   5ug/ml     se17 81-90 (439+-77/1000ms)
   1ug/ml     se17 91-100 (127+-23/1000ms)
   0.5ug/ml    se17 101-110 (110+-19/1000ms)
   20ug/ml    se18 1-10 (482+-51/1000ms)
   5ug/ml     se18 11-20 (426+-52/1000ms)
   1ug/ml     se18 21-30 (155+-7/1000ms)
   0.1ug/ml    se18 31-40 (13+-4/1000ms)

Sep19th96  Effect of Cell Density
   Ecta 15ul packed in 300ul (25:500)
   FMID 2ul packed in 1ml (1:500)
   F-B18 1ug/ml, 1:500   se19 1-10 (123+-18/1000ms)
   F-B18 1ug/ml, 1:500   se19 11-20 (178+-13/1000ms)
   F-B18 1ug/ml, 1:500   se19 21-30 (179+-31/1000ms)
   F-B18 1ug/ml, 25:500   se19 31-41(32+-5/1000ms)!!

Sep20th96  F-B18 Isotherm Cell Dil 25:500 \f-b18\
   100ug/ml    se20 1-10 (486+-47/1000ms)
   50ug/ml    se20 11-20 (548+-71/1000ms)
   20ug/ml    se20 21-30 (413+-53/1000ms)
   10ug/ml    se20 31-40 (217+-39/1000ms)
   5ug/ml     se20 41-50 (110+-26/1000ms)
   F-B18 1ug/ml, 25:500   se19 31-41(32+-5/1000ms)

Sep24th96  F-B18 20ug/ml (1:500)   \f-b18\se24 1-40
   F-B18 0.5ug/ml (1:500)  \f-b18\se24 41-70
Sep25th96  F-B18 Mu's
Sep26th96  F-B18 20ug/ml    \f-b18\se26 6-10, 11-37

Sep30th96 * CHIP (2ul + 8ul)   \chip\se30 1-33  (with times)
   CHIP + B18 20ug/ml   \chip\se30 34-43  (with times)
Oct1st96 * CHIP     \chip\oc01 1-50  (Short times ~20sec)
  * CHIP + B18 20ug/ml   \chip\oc01 51-98  (Short times ~20sec)

Nov25th96  Bottom Frac RBC ema   no25 1-40
   Top Frac ema    no25 41_80
   Middle Frac ??
Dec4th96  >27 Dense, ema   de04 1-40
   ema RBC    de04 41-80
Dec5th96  >27 Dense, ema   \hemden\ema\de05 1-40
   ema (same pipe)   \ema\de05 41-80
Jan16th97 * rhph     \rhph\ja16 1-30
        \rhph\ja16 31-60
Jan17th97 * rhph     \rhph\ja17 21-40 Rp=1.66+-0.07um
Jan22nd97 * rhph     \rhph\ja22 1-30, 31-40, 41,50

DISK

mr13th97  ema+dids    \dids\mr13 1-20 UV Excitation yeild EMA>>DIDS!
mr18th97  f-albumin 43kD incorrporation  \f-alb\mr18 1-10
   45.6kD Ankyrin Fragment  \ank-frag\mr18 11-37
  * f-albumin    \f-alb\mr18 38-47 0.5mg/ml, 1978±560c/1000ms Tounge Fluorescence at constant Rp
  * f-albumin    \f-alb\18 48-60 1.0mg/ml 7870±1423c/1000ms
  * f-albumin    \f-alb\18 61-701.5mg/ml 6756±3205c/1000ms
  * f-albumin    \f-alb\18 71-802.0mg/ml 14,318±2870c/1000ms
  * f-albumin    \f-alb\18 81-90 7.3mg/ml 24,171±10,134c/1000ms
  * f-albumin    \f-alb\18 91-100 94.5mg/ml 9,032±1304c/1000ms ?? Incomplete lysis!!

  * f-anky-frag    \ank-frag\mr18 101-110 0.5mg/ml 47±5c/1000ms Well Intensity Incomplete Lysis!!
  * f-anky-frag    \ank-frag\mr18 111-120 1.0mg/ml 82±20c/1000ms, Complete Lysis
  * f-anky-frag    \ank-frag\mr18 121-130 1.5mg/ml 101±23c/1000ms, Complete Lysis, No apparent Fragmemtation
  * f-anky-frag    \ank-frag\mr18 131-140 2.0mg/ml 93±18c/1000ms, Incomplete Lysis Normal Morphology
  * f-anky-frag    \ank-frag\mr18 141-150 3.1mg/ml 171±35c/1000ms
  * f-anky-frag    \ank-frag\mr18 151-160 10.2mg/ml 588±49c/1000ms, Complete Lysis

mr19th97 * f-anky-frag    \ank-frag\mr19 1-10 6mg/ml 60±7c/1000ms, Complete Lysis
  * f-anky-frag    \ank-frag\mr19 11-20 10mg/ml 935±209c/1000ms, Complete Lysis
  * f-anky-frag    \ank-frag\mr19 21-30 30 mg/ml 1600±208c/1000ms
  * f-anky-frag    \ank-frag\mr19 31-50 60mg/ml 1668±545c/1000ms, Fragmentation & Invaginations
  * f-anky-frag    \ank-frag\mr19 51-60 100mg/ml 1111±230c/1000ms, All Spherocytes

mr21st97 * ema/resealed ghost control  \ema\mr21 1-20

   dids (Mol Pro), 1hr@37°, 50uM  \dids\mr21 21-25, Realized the UV DIDS quantum yeild is lower than that of UV EMA
   dids (Mol Pro), 1hr@37°, 100uM \dids\mr21 26-30
   dids (Sigma), 1hr@37°, 50uM  \dids\mr21 31-35
   dids (Sigma), 1hr@37°, 100uM  \dids\mr21 36-40

  * Ank Frag + EMA, 0.429mg/ml  \ema\ank\mr21 41-50
  * Ank Frag + EMA, 0.514mg/ml  \ema\ank\mr21 51-60
   Ank Frag + EMA, 0.686mg/ml  \ema\ank\mr21 61-66 Vesiculation, invaginations
   Ank Frag + EMA, 1.7mg/ml  \ema\ank\mr21 71-73 Unpipetteable fluorescent fragments

mr23rd97 * EMA + DIDS @ 100uM,1hr, 37°  \ema\dids\mr23 1-59 UV-even, blue-odd (NB: UV excites EMA)
  *     EMA, ghost reseal   \ema\mr23 61-100
  * EMA+Anf Frag    \ema\ank\mr23 101-120, 300ug/ml
  * EMA+Anf Frag    \ema\ank\mr23 121-140, 400ug/ml
  * EMA+Anf Frag    \ema\ank\mr23 141-160, 500ug/ml
   EMA+Anf Frag    \ema\ank\mr23 161-180, 600ug/ml Fragmentation

mr24th97 * DIDS 100uM, 24hr @ 37°  \dids\mr24 1-30
mr25th97  DIDS + antibiotic   \dids\mr25 1-3 No Signal + 1 fluorescent dot per cell??

mr26th97 * DIDS 100uM 12hrs@ 37°  \dids\mr26 1-34

   EMA + Ank Frag   \ema\mr26 40-60 0ug/ml
   EMA + Ank Frag   \ema\ank\mr26 61-80 400ug/ml
  * EMA + Ank Frag   \ema\ank\mr26 81-100 800ug/ml, Spherocytes deflated for aspiration.

mr27th97  DIDS 100uM 24hrs@ 37°  \dids\mr27 1-30
mr28th97 * DIDS 100uM 1hr@ 37°   \dids\mr28 1-40

my28th97 * anti-CHIP APQ1   \chip\my28 1-44, Measuring cap collection times
my29th97 * anti-CHIP APQ1   \chip\my29 1-30, Measuring cap collection times (Syquest Disk Problem, some files lost)

Oct18th97 * Rh_Null - EMA    \rh-null\ema\ oc18 1-40
  * Control (Rh_Null) EMA   \ema\ oc18 41-80
Oct20th97 * Rh_Null + fl_anti_Rh50  \rh_null\a_rh50\ oc20 1-10 Signal (well) 13.7±2.4 /1000ms
  * Ctrl  + fl_anti_Rh50   \a_rh50\ oc20 11-50  Signal (well) 63.2±8.6 /1000ms images 11-20
  * Rh_Null + EMA   \rh_null\ema\ oc20 51-90
   Rh_Null + fl anti RhD       No Signal
  * Ctrl + fl anti RhD   \a_rhd\ oc20 91-130  Signal 23±6 /1000ms

Dec 4th 1997 * DIDS <30min    de04 1-30   8min 100µM DIDS incubation, wash
  * DIDS 2hrs @ 37°   de04 31-52   8min 100µM DIDS incubation, wash, 2hrs @ 37°
Jan22nd98 * DIDS no 37° incubation   ja22 1-30   6min 100µM DIDS incubation, wash, images 20-60min
  * DIDS 1hr 37° Incubate   ja22 31-60   10min 100µM DIDS incubate, wash, 1hr 37° incubate
Jan26th98 * DIDS 2hr 37° Incubate   ja26 1-10    10min 100µM DIDS incubate, wash, 2hr 37° incubate
May6th98  anti-spectrin (Stanley Schrier)      FMA & RhPh Double Label
   anti-spectrin polyclonal + serum proteins @ 14mg/ml. Assume! the 14mg/ml of polyclonal has1mg/ml of anti-spectrin*
   0.5mg/ml* resulted in fragmented vesicles only (Control was discocyte) (5µl packed FMA RBC + 10µl Lysis/ATP + 10µl anti-sp)
   0.2mg/ml* resulted in discocytes (5µl packed FMA RBC + 16µl Lysis/ATP + 4µl anti-sp) NO Rigidity Increase by Mu & Obvious Flicker !
   Noticable increased fragility of cells which form projections, as if the skeleton is falling apart. See tape sequence after Mu's.
   Mu Sequence: Tape May6th1998: 7:32:15 up buckle soft, 7:34:56 up/dn soft ,double label ok: 7:36:40 up/dn soft.

May8th98  anti-spectrin
   Reseal scale up for Mu and Ectacytometry. Without fluorescent labeling
   Control  [200µl Lysis/ATP Buffer + 50µl Packed RBC]
   Anti-Sp @ 160µg/ml  [160µl Lysis/ATP + 40µl Anti-Sp (@ 1mg/ml*) + 50µl Packed RBC]
   40µl of packed ghosts gave very low noise compared to high spin wash before reseal.
   Anti-sp gave small detectable rigidity increase.
   Mu [tape May6th98]
   Anti-SP 1:45:57, 1:49:27, 1:54:42, 2:26:39, 2:30:07, 2:35:48, 2:40:40, 2:52:00, 2:55:29, 2:58:45
   Control  3:57:12, 4:03:07, 4:10:59, 4:14:49, 4:16:51, 4:20:21

May14th98  anti-spectrin @ 400µg/ml, Dialized stock measured @1mg/ml*, For ectacytometer try just 10µl of packed RBC's
   Control  [ 40µl Lysis/ATP/RhPh + 10µl Packed FMA RBC]
   Anti-sp 400µg/ml [20µl anti-sp + 20µl lysis/ATP/RhPh + 10µl Packed FMA RBC] + 20min + 5µl 10xPBS + 30min @ 37C
   Anti-Rabbit IgG 400µg/ml  [48µl Lysis/ATP/RhPh + 2µl antiRabbit @ 10mg/ml + 10µl Packed FMA RBC]
   Both the control and non-specific Anti-Rabbit gave normal morphjology and normal ecta signal (10µl of Packed Ghosts works fine)
   Anti-sp again gave fragments and unworkable morphologies, see 1:03:00 for normals, anti-sp & anti-rabbit [tapeMay6th98]
   Anti-sp looked very rigid/flat in ecta (which of course it was not)

May21st98  Fluorescein- Anti Rabbit IgG @ 400µg/ml My21 - 1-10 (Odd F-IgG, Even RhPh) Morphology etc normal